foxm1 in breast cancer and drug resistance€¦ · for the degree of doctor of philosophy...
TRANSCRIPT
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FOXM1 in breast cancer and
drug resistance
Thesis submitted by
Julie Millour
To Imperial College London
For the degree of Doctor of Philosophy
Department of Surgery and Cancer 8th floor MRC Cyclotron Building
Imperial College Hammersmith Hospital
Du Cane Road London W12 0NN
2012
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DECLARATION OF ORIGINALITY
Unless otherwise stated in text, this thesis is the result of my own work.
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ABSTRACT
Endocrine agents have become the primary adjuvant treatment for breast cancer. In
addition to endocrine therapy, cytotoxic chemotherapeutic agents have also been
frequently used in the neoadjuvant and adjuvant settings, to reduce tumour size prior
to surgery or to reduce the chance of relapse or metastasis. However, patients can
be resistant to endocrine and chemotherapeutic agents, or become resistant after
long term treatment. In this study, I investigated the role and the regulation of FOXM1
in the sensitivity and resistance to the endocrine agent, tamoxifen, and the cytotoxic
chemotherapeutic agent, epirubicin. Firstly, I demonstrated that tamoxifen repressed
FOXM1 expression in sensitive but not in tamoxifen resistant breast cancer cell lines.
In MCF-7 cells, FOXM1 protein and mRNA expression levels were regulated by ER-
ligands, and depletion of ERα by RNA interference down-regulated FOXM1
expression. Importantly, ectopic expression of FOXM1 abrogated the cell cycle arrest
mediated by the anti-oestrogen tamoxifen, and conferred tamoxifen resistance to
MCF-7 cells. In contrast, silencing of FOXM1 in tamoxifen resistant cells abolished
oestrogen-induced MCF-7 cell proliferation and overcame acquired tamoxifen
resistance. Secondly, FOXM1 expression analysis in epirubicin resistant MCF-7 cells
showed a higher level compared with MCF-7 cells. In addition, epirubicin treatment
down-regulated FOXM1 expression in MCF-7, but FOXM1 protein level remained
constant in epirubicin resistant MCF-7 cells. I established that p53 repressed FOXM1
expression in MCF-7 cells, while this protein is lost in the MCF-7 epirubicin resistant
cells. I also found that ataxia-telangiectasia mutated (ATM) was overexpressed at
protein and mRNA levels in epirubicin resistant MCF-7 compared with MCF-7 cells,
and that ATM depletion strongly decreased FOXM1 expression. Epirubicin treatment
increased DNA damage levels in MCF-7 cells while this remained constant in
similarly treated epirubicin resistant MCF-7 cells, suggesting a higher level of DNA
repair in these cells. Taken together, these results indicate that deregulation of
FOXM1 may contribute to resistance to endocrine and cytotoxic agents through its
involvement in cell proliferation and DNA repair.
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PUBLICATIONS Down CF, Millour J, Lam EW, Watson RJ. Biochim Biophys Acta. 2012 Mar 30.
Binding of Foxm1 to G2/M gene promoters is dependent upon B-Myb.
Horimoto Y, Hartman J, Millour J, Pollock S, Olmos Y, Ho KK, Coombes RC,
Poutanen M, Mäkelä SI, El-Bahrawy M, Speirs V, Lam EW.Am J Pathol. 2011 Jul 13.
ERβ1 Represses FOXM1 Expression through Targeting ERα to Control Cell
Proliferation in Breast Cancer.
Millour J, de Olano N, Horimoto Y, Monteiro LJ, Langer JK, Aligue R, Hajji N, Lam
EW. Mol Cancer Ther. 2011 Jun;10(6):1046-58. Epub 2011 Apr 25. ATM and p53
regulate FOXM1 expression via E2F in breast cancer epirubicin treatment and
resistance.
Chen J, Gomes AR, Monteiro LJ, Wong SY, Wu LH, Ng TT, Karadedou CT, Millour
J, Ip YC, Cheung YN, Sunters A, Chan KY, Lam EW, Khoo US. PLoS One. 2010 Aug
20;5(8):e12293. Constitutively nuclear FOXO3a localization predicts poor survival
and promotes Akt phosphorylation in breast cancer.
Millour J, Constantinidou D, Stavropoulou AV, Wilson MS, Myatt SS, Kwok JM,
Sivanandan K, Coombes RC, Medema RH, Hartman J, Lykkesfeldt AE, Lam EW.
Oncogene. 2010 Mar 8. FOXM1 is a transcriptional target of ERalpha and has a
critical role in breast cancer endocrine sensitivity and resistance.
Kwok JM, Peck B, Monteiro LJ, Schwenen HDC, Millour J, Coombes RC,Myatt SS,
Lam EW. Mol Cancer Res 2010; 8(1):24-34 FOXM1 confers acquired Cisplatin
resistance in Breast Cancer cells.
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ACKNOWLEDGMENTS First, I would like to express my gratitude to my supervisor Prof. Eric Lam for giving
me the opportunity to undertake my PhD in his laboratory in London; it was a crucial
step in driving my career choice.
I also would like to thank all members of the lab. Among them, previous lab
members who have been very helpful in supporting me to develop ideas, those with
whom I spent most of these 3 years including week-ends for giving me technical and
personal support, and the new lab members for bringing new and fresh atmosphere
into the lab.
Last but not the least; I would like to thank my mother for supporting me on the phone
from France, my friends and my boyfriend for supporting me through good and bad
periods and always encouraging me throughout my PhD.
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TABLE OF CONTENTS
DECLARATION OF ORIGINALITY ........................................................................................... 2
ABSTRACT ............................................................................................................................. 3
PUBLICATIONS...................................................................................................................... 4
ACKNOWLEDGMENTS ........................................................................................................... 5
TABLE OF CONTENTS ............................................................................................................ 6
LIST OF TABLES ...................................................................................................................11
LIST OF TABLES ...................................................................................................................13
ABBREVIATIONS ...................................................................................................................14
CHAPTER 1 INTRODUCTION ................................................................................................16
1.1 BREAST CANCER ....................................................................................................17
1.1.1 Epidemiology ...............................................................................................................17
1.1.2 Breast cancer development .........................................................................................19
1.2 BREAST CANCER CLINICAL MANAGEMENT ..............................................................19
1.2.1 Breast cancer chemotherapies ....................................................................................22
1.2.1.1 DNA damage agents ................................................................................................22
1.2.1.2 DNA damage response pathways.............................................................................25
1.2.2 Breast cancer endocrine therapies ..............................................................................29
1.2.2.1 Anti-oestrogen therapies ...........................................................................................29
1.2.2.2 The oestrogen receptor pathway ..............................................................................31
1.3 GENETIC AND NEW THERAPIES .................................................................................34
1.3.1 Genetic predispositions and mutations ........................................................................34
1.3.2 Targeted therapies ......................................................................................................35
1.4 BREAST CANCER RECURRENCE ...............................................................................37
1.4.1 Recurrence of the disease ...........................................................................................37
1.4.2 Chemotherapy resistance ............................................................................................38
1.4.3 Anti-oestrogen therapy resistance ...............................................................................39
1.4.4 Targeted therapy resistance ........................................................................................41
1.4.5 Potential strategies overcoming drug resistance ..........................................................43
1.5 FORKHEAD BOX TRANSCRIPTION FACTORS ...........................................................46
1.6 FORKHEAD BOX M1 (FOXM1) ......................................................................................46
1.6.1 Structure ......................................................................................................................46
1.6.2 Regulation ...................................................................................................................47
1.6.3 FOXM1 function...........................................................................................................50
1.6.3.1 Cell cycle ..................................................................................................................50
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1.6.3.2 Regenerative cell proliferation ..................................................................................50
1.6.3.3 Senescence ..............................................................................................................51
1.6.3.4 Apoptosis ..................................................................................................................51
1.6.3.5 DNA damage ............................................................................................................51
1.6.3.6 Angiogenesis ............................................................................................................52
1.7 FOXM1 IN CANCER ......................................................................................................53
1.7.1 FOXM1 in breast cancer ..............................................................................................54
1.7.2 Development of FOXM1 inhibitors ...............................................................................55
1.8 HYPOTHESES AND OBJECTIVES: FOXM1 as a therapeutic strategy to overcome drug
resistance .............................................................................................................................56
1.8.1 FOXM1 regulation and role in tamoxifen sensitivity and resistance..............................58
1.8.2 FOXM1 regulation and role in chemotherapy sensitivity and resistance ......................58
CHAPTER 2 MATERIAL AND METHODS ................................................................................60
2.1 CELL CULTURE ............................................................................................................61
2.1.1 Cell lines ......................................................................................................................61
2.1.2 Stably transfected cell lines .........................................................................................61
2.1.3 Knock-out cells ............................................................................................................61
2.1.4 Drug resistant cell lines ................................................................................................62
2.1.5 Cell line maintenance ..................................................................................................63
2.1.6 Chemicals ....................................................................................................................63
2.2 PROTEIN ANALYSIS .....................................................................................................64
2.2.1 Preparation of total protein lysates and determination of protein concentration ...........64
2.2.2 Western blotting or sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-
PAGE) ..................................................................................................................................64
2.3 PULL-DOWN using biotin-labelled oligonucleotides .......................................................67
2.4 IMMUNOPRECIPITATION AND IMMUNOBLOTTING ...................................................67
2.5 CHROMATIN IMMUNOPRECIPITATION (ChIP) ............................................................68
2.5.1 Beads preparation .......................................................................................................68
2.5.2 Cells preparation .........................................................................................................68
2.5.3 Sonication ....................................................................................................................69
2.5.4 DNA/beads-antibody incubation ..................................................................................69
2.5.5 DNA elution, purification and Polymerase Chain Reaction (PCR) ................................69
2.5.6 DNA gel electrophoresis ..............................................................................................71
2.6 RNA ANALYSIS .............................................................................................................71
2.6.1 Total RNA extraction ...................................................................................................71
2.6.2 First strand cDNA synthesis.........................................................................................72
2.6.3 Primers ........................................................................................................................72
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2.6.4 Real-time quantitative PCR (RT-qPCR) .......................................................................74
2.7 DNA MANIPULATION ....................................................................................................74
2.7.1 Plasmid amplification and extraction ............................................................................74
2.7.2 DNA mutation and sequencing ....................................................................................75
2.7.3 Plasmid DNA transfection ............................................................................................76
2.7.4 Luciferase assay ..........................................................................................................77
2.7.5 Host cell reactivation assay (HCR) ..............................................................................78
2.8 RNA INTERFERENCE ...................................................................................................80
2.8 IMMUNOFLUORESCENCE MICROSCOPY ..................................................................80
2.9 SRB assay......................................................................................................................81
2.10 CELL CYCLE ANALYSIS .............................................................................................82
2.11 STATISTICAL ANALYSIS ............................................................................................82
CHAPTER 3 FOXM1 is a transcriptional target of ERalpha and has a critical role in breast cancer
endocrine sensitivity and resistance ..........................................................................................83
3.1 Introduction.....................................................................................................................84
3.2 Results ...........................................................................................................................85
3.2.1 Transcriptional regulation of FOXM1 by ERα in endocrine sensitive breast cancer cells
.............................................................................................................................................85
3.2.1.1 ERα ligands and ERα silencing modulate FOXM1 expression ..................................85
3.2.1.2 FOXM1 promoter responds to ERα ligands ..............................................................91
3.2.1.3 ERα and HDAC2 bind on the ERE-like site of FOXM1 promoter in vitro ...................93
3.2.1.4 ERα binds specifically to FOXM1 promoter in vivo ....................................................95
3.2.1.5 FOXM1 silencing is cytotoxic for MCF-7 cells independent of the E2 mitogenic effect
.............................................................................................................................................97
3.2.2 Deregulation of FOXM1 in tamoxifen resistant breast cancer cells ..............................99
3.2.2.1 Deregulation of FOXM1 protein and mRNA expression in tamoxifen resistant cells ..99
3.2.2.2 Reduced G1 cell cycle arrest in tamoxifen resistant cells after OHT ....................... 102
3.2.2.3 Combination of OHT and FOXM1 silencing has a cytostatic effect on MCF-7
tamoxifen resistant cells ..................................................................................................... 105
3.2.3 Potential mechanisms of tamoxifen resistance .......................................................... 107
3.2.3.1 FOXM1 phosphorylation and transcriptional activation ........................................... 107
3.2.3.2 ERα overexpression and silencing do not alter FOXM1 expression in tamoxifen
resistant cells ..................................................................................................................... 109
3.2.3.3 Protein deregulations in tamoxifen resistant cells ................................................... 110
3.3 Discussion .................................................................................................................... 112
3.3.1 Regulation of ER and FOXM1 through a positive feedback loop in breast cancer cells
........................................................................................................................................... 112
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3.3.2 Uncoupled ER and FOXM1 feedback loop regulation in tamoxifen resistant breast
cancer cells ........................................................................................................................ 113
3.3.3 Deregulated AIB1, an ERα co-factor, in tamoxifen resistant breast cancer cells ........ 114
3.3.4 Deregulation of FOXM1 negative and positive regulators as potential mechanisms of
tamoxifen resistance .......................................................................................................... 115
3.3.5 Conclusion ................................................................................................................. 117
3.4 Future work .................................................................................................................. 118
CHAPTER 4 ATM and p53 regulate FOXM1 expression via E2F in breast cancer epirubicin treatment
and resistance ...................................................................................................................... 120
4.1 Introduction................................................................................................................... 121
4.2 Transcriptional regulation of FOXM1 by p53 in epirubicin sensitive MCF-7 cells .......... 122
4.2.1 Activation of p53 transcriptionally represses FOXM1 ................................................. 122
4.2.2 p53 can regulate FOXM1 through an E2F site in its promoter.................................... 127
4.3 Differential mechanism of FOXM1 regulation in epirubicin resistant MCF-7 cells .......... 132
4.3.1 Deregulation of FOXM1 protein and mRNA levels in epirubicin resistant cells ........... 132
4.3.2 Increased DNA repair in epirubicin resistant cells ...................................................... 133
4.3.3 ATM is involved in FOXM1 regulation and epirubicin resistance ................................ 138
4.4 Discussion .................................................................................................................... 146
4.4.1 FOXM1 is a crucial target of p53 ............................................................................... 146
4.4.2 p53 status is not a determinant of epirubicin response .............................................. 147
4.4.3 FOXM1 is a target of ATM ......................................................................................... 148
4.4.4 FOXM1 involvement in DNA repair and cell survival .................................................. 149
4.4.5 Conclusion ................................................................................................................. 150
4.5 Future work .................................................................................................................. 152
5.1 Introduction................................................................................................................... 154
5.2 FOXM1 is essential for DNA repair in epirubicin resistant breast cancer cells .............. 155
5.3 Enhanced recruitment of FOXM1 and P-H2AX in MCF-7EPIR cells following DNA breaks
........................................................................................................................................... 158
5.4 FOXM1 is required for the activation of ATM, H2AX and CHK2 DNA repair proteins .... 161
5.5 FOXM1 is required for ATM auto-phosphorylation upon epirubicin ............................... 166
5.6 Transcriptional regulation of NBS1 by FOXM1 ............................................................. 168
5.7 NBS1 mediates ATM activation upon epirubicin ........................................................... 171
5.8 Discussion .................................................................................................................... 172
5.8.1 FOXM1 is involved in ATM and its downstream substrates phosphorylations ............ 172
5.8.2 FOXM1 regulates NBS1 ............................................................................................ 173
5.8.3 NBS1 activates ATM auto-phosphorylation ................................................................ 174
5.8.4 FOXM1 function in DNA repair .................................................................................. 174
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5.8.5 Conclusion ................................................................................................................. 176
5.9 Future work .................................................................................................................. 178
CHAPTER 6 FINAL DISCUSSION ......................................................................................... 179
CHAPTER 7 SUPPLEMENTAL DATA .................................................................................... 185
REFERENCES ..................................................................................................................... 188
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LIST OF TABLES
Figure 1.1 Number of deaths and ages-specific mortality rates of breast cancer, UK, 2008.
Cancer Research UK............................................................................................................18
Figure 1.2 Two main types of breast cancer and therapies. ..................................................21
Figure 1.3 Targets for chemotherapies .................................................................................24
Figure 1.4 Cell cycle checkpoints following DNA damage. ...................................................26
Figure 1.5 Double strand break repair. .................................................................................28
Figure 1.6 Effects of anti-oestrogens. ...................................................................................30
Figure 1.7 Direct interactions of oestrogen receptor with its cofactors. ...............................32
Figure 1.8 HER2 signalling and pathway targeted therapy ...................................................36
Figure 1.9 FOXM1 structure. ................................................................................................47
Figure 1.10 Cell cycle-dependent phosphorylation of FOXM1. Cell cycle-dependent
regulation of FoxM1. .............................................................................................................49
Figure 1.11 FOXM1 functions. ..............................................................................................53
Figure 2.1 DSB detection and repair model. .........................................................................70
Figure 2.2 Host Cell Reactivation. ........................................................................................79
Figure 3.1 Expression of FOXM1 and ERα in response to E2, tamoxifen and ICI treatments
in breast cancer cell lines .....................................................................................................88
Figure 3.2 Induction of FOXM1 expression by E2 is antagonized by OHT and ICI in MCF-7
cells. .....................................................................................................................................89
Figure 3.3 Effects of ERα silencing on the expression of FOXM1. ........................................90
Figure 3.4 ERα induces the transcriptional activity of the human FOXM1 gene through an
ERE-like site. ........................................................................................................................92
Figure 3.5 ERα binds directly to the ERE-like site on FOXM1 promoter in vitro ....................94
Figure 3.6 Chromatin immunoprecipitation (ChIP) analysis of the human FOXM1 promoter.96
Figure 3.7 Effects of FOXM1 silencing on E2-induced proliferation of MCF-7 cells. ..............98
Figure 3.8 Full length and partial FOXM1 overexpression reduced the downregulation of
FOXM1 and its target genes following tamoxifen treatment ................................................ 101
Figure 3.9 Cell cycle regulation in wild-type (MCF-7), tamoxifen resistant (MCF-7TAMR4) and
constitutively active ∆N-FOXM1 expressing MCF-7 cells in response to tamoxifen treatment.
MCF-7, MCF-7 TAMR4 and MCF-7 ∆N-FOXM1 cells were treated with 10-6 mol/L of OHT in a
time course of 72 h. ............................................................................................................ 103
Figure 3.10 FOXM1 upregulation rescues OHT-induced cell growth arrest and decrease of
endogenous FOXM1 in response to tamoxifen treatment. .................................................. 104
Figure 3.11 FOXM1 silencing rescues tamoxifen anti-growth effect in MCF-7TAMR4 cells.. 106
Figure 3.12 Constitutively active ∆N-FOXM1 expressing MCF-7 cells show the same protein
expression pattern as MCF-7TAMR4 cells in response to tamoxifen treatment. ................... 108
Figure 3.13 ERα ectopic expression and silencing in the tamoxifen resistant ERα negative
MDA-MB-231 and ERα positive MCF-7TAMR4 breast cancer cells ..................................... 109
Figure 3.14 AIB-1 pattern expression in tamoxifen sensitive and resistant MCF-7 cells. Both
cell lines were treated with OHT over 72 h. ........................................................................ 111
Figure 3.15 Chromatin immunoprecipitation of ERβ and ERα in MCF-7 cells. .................... 112
Figure 3.16 Potential pathways in endocrine therapy ......................................................... 117
Figure 4.1 Expression of FOXM1 in response to epirubicin treatment in breast cancer cell
lines .................................................................................................................................... 124
Figure 4.2 Activation of p53 in MCF-7 cells represses FOXM1 expression ......................... 125
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Figure 4.3 FOXM1 repression by p53 in a p21-independent manner .................................. 126
Figure 4.4 E2F1 is decreased in response to epirubicin in MCF-7 cells .............................. 129
Figure 4.5 FOXM1 promoter activity in response to epirubicin. ........................................... 130
Figure 4.6 Modulation of FOXM1 promoter by p53 and E2F1 via E2F binding site ............. 131
Figure 4.7 Characterisation of epirubicin resistant MCF-7EPIR cells. .................................. 134
Figure 4.8 Inverse correlation between FOXM1 and p53 expression in MCF-7 and MCF-
7EPIR cell lines ................................................................................................................... 135
Figure 4.9 Epirubicin resistant MCF-7EPIR cells show a reduction of DNA damage in
response to epirubicin treatment ........................................................................................ 136
Figure 4.10 Increased expression of ATM in epirubicin resistant MCF-7EPIR cells. ............ 137
Figure 4.11 ATM inhibition re-sensitises MCF-7EPIR cells to FOXM1 downregulation
epirubicin-induced .............................................................................................................. 141
Figure 4.12 ATM is involved in FOXM1 regulation in epirubicin resistant MCF-7EPIR cells. 142
Figure 4.13 Phosphorylation and stabilisation of FOXM1 in MCF-7EPIR cells. .................... 143
Figure 4.14 E2F1 occupancy on FOXM1 promoter remains steady in MCF-7EPIR cells. .... 144
Figure 4.15 Silencing of FOXM1 combined with epirubicin treatment increases cell death in
MCF-7EPIR cells. ................................................................................................................ 145
Figure 5.1 FOXM1 depletion alters DNA repair in MCF-7EPIR
cells .................................... 157
Figure 5.2 Increased in the recruitment of FOXM1 and repair factors at DNA breaks in MCF-
7EPIR
cells .......................................................................................................................... 160
Figure 5.3 FOXM1 silencing reduces ATM phosphorylation on serine 1981 in MCF-7EPIR
cells. ................................................................................................................................... 162
Figure 5.4 FOXM1 silencing decreases H2AX phosphorylation on serine 139 in MCF-7EPIR
cells.. .................................................................................................................................. 163
Figure 5.5 FOXM1 silencing in human fibroblast cells abrogates ATM phosphorylation. .... 164
Figure 5.6 FOXM1 silencing in human fibroblast cells decreases Chk2 and H2AX
phosphorylation .................................................................................................................. 165
Figure 5.7 FOXM1 does not regulate ATM transcriptionally. ............................................... 167
Figure 5.8 FOXM1 silencing significantly decreases NBS1 mRNA levels. .......................... 169
Figure 5.9 FOXM1 binds directly to NBS1 promoter through the Forkhead binding site (FHK).
........................................................................................................................................... 170
Figure 5.10 NBS1 is required for ATM activation upon epirubicin ....................................... 171
Figure 5.11 Differential pathways upon epirubicin in sensitive and resistant MCF-7 cells. .. 177
Figure 6.1 Thiostrepton as a potential candidate to overcome endocrine and chemotherapy
resistance in breast cancer. ................................................................................................ 184
Figure S.D.7.1. Schematic representation of the Apa I FOXM1 construct, showing the wild-
type ERE, and three mutants ERE (mERE) sequences (mutant analysed by Demetra
Constantinidou). ................................................................................................................. 186
Figure S.D.7.2. ERα induces the transcriptional activity of the human FOXM1 gene through
an ERE proximal to the transcription start site (experiment performed by Demetra
Constantinidou). ................................................................................................................. 186
Figure S.D.7.3 Ectopic expression of FOXM1 reduces MCF-7 cells sensitivity to cell death
(Experiment performed by Julia K. Langer). ....................................................................... 187
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LIST OF TABLES
Table 2.1 SDS-PAGE and ChIP buffers ................................. Error! Bookmark not defined.
Table 2.2 Antibodies for western blotting and ChIP ................ Error! Bookmark not defined.
Table 2.3 DNA gel electrophoresis buffers ............................. Error! Bookmark not defined.
Table 2.4 Human and mouse gene-specific primer pairs for RT-qPCR and ChIP ........... Error!
Bookmark not defined.
Table 2.5 Bacterial culture reagents ....................................... Error! Bookmark not defined.
Table 2.6 Expression vectors ................................................. Error! Bookmark not defined.
Table 2.7 Promoter constructs ................................................ Error! Bookmark not defined.
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ABBREVIATIONS
5-FU 5-fluorouracil ABC-transporters ATP-binding cassette transporters AF Activation function AI Aromatase inhibitor ARF Alternative Reading Frame ATM Ataxia telangiectasia mutated ATR ATM and Rad3-related BRCA1 Breast cancer susceptibility gene 1 BSA Bovine Serum Albumin CDK Cyclin dependent kinase DNA Deoxyribonucleic acid cDNAs Complementary deoxyribonucleic acid CHK2 Checkpoint kinase 2 CKI Cyclin dependent kinase inhibitors DBD DNA binding domain DMEM Dulbecco.’s Modified Eagle.’s Medium DMSO Dimethyl sulphoxide DNA-PK DNA-dependent protein kinase dNTP Di-nucleotide triphosphate DSB DNA double strand break E2 Estradiol EDTA Ethylenediaminetetraacetic acid EGFR Epidermal growth factor receptor ER Oestrogen receptor ERAP ER-interacting proteins ERE Oestrogen response element ERK Extracellular signal-regulated kinase FCS Foetal Calf Serum HAT Histone acetyl transferase HDAC Histone deacetylase HER2 Human epidermal growth factor receptor HR Homologous recombination ICI or ICI182780 Fulvestrant IGFR Insulin-like growth factor IR Ionizing radiation LBD Ligand-binding domain M Mitotic phase MAPK Mitogen-activated protein kinase MDR Multidrug resistance MEF Mouse embryonic fibroblasts MMP Matrix metalloproteinase MnSOD Manganese superoxide dismutase MRP1 Multidrug resistance-associated protein 1 MUC4 Membrane-associated glycoprotein mucin-4 NBS1 Nijmegen breakage syndrome NcoR Nuclear receptor co-repressor
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NHEJ Non-homologous end-joining NSC Non-specific control OHT Tamoxifen PARP1 Poly (ADP-ribose) polymerase 1 PBS Phosphate Buffered Saline PGP P-glycoprotein PI Propidium iodide PI3K Phosphatidylinositol 3 kinases PIK3CA Phosphoinositol 3-kinase catalytic unit PIP2 3,4,5-triphosphate PIP3 Phosphatidylinositol 4,5-biphosphoate PLK1 Polo-like kinase 1 PR Progesterone receptor pRB Phosphorylated retinoblastoma protein PTEN Phosphatase and tensin homologue RB Retinoblastoma protein RIP Receptor interacting proteins RNA Ribonucleic acid ROS Reactive oxygen species RT Room temperature S Synthesis phase SERD Selective ERα down-regulators SERM Selective ERα modulator SMC1 Structural maintenance of chromosome protein 1 SMRT Silencing mediator for retinoid and thyroid hormone
receptors TBP TATA-binding protein TEMED Tetramethylethylenediamine TK Tyrosine kinases TKI Tyrosine kinase inhibitor uPA Urokinase-type kinase plasminogen activator uPAR Urokinase-type kinase plasminogen activator receptor UV Ultraviolet VEGF Vascular endothelial growth factor XRCC4 X-ray repair cross-complementing protein 4
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CHAPTER 1 INTRODUCTION
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1.1 BREAST CANCER
The development of cancer or carcinogenesis is a process by which normal
cells transform into cancer cells. It is characterised by a series of alterations
occurring at genetic and cellular levels leading to uncontrolled cell division and
consequently formation of a malignant mass. These alterations affect two types of
genes: oncogenes and tumour suppressors. An oncogene is a gene involved in cell
proliferation and differentiation signalling pathways, which has been modified by
mutation or in a post-translational manner, and consequently has acquired potential
to cause cancer. In contrast to oncogenes, tumour suppressors are anti-oncogenic
and protect cells from deregulation by inhibiting cell growth and inducing cell death.
Hanahan and Weinberg summarize all biological properties acquired by tumour cells
as self-sufficiency in growth signals, loss of sensitivity to anti-growth signals, loss of
apoptotic and senescence capacities, acquisition of sustained angiogenesis and
invaded neighbouring tissues (Hanahan and Weinberg 2011). Cancer is now the
leading cause of death in the UK (Cancer Research UK) and around 293,000 people
are diagnosed every year in the UK. There are about 200 different types of cancer
but four of them, breast, lung, colorectal and prostate, account for over half of all new
cases.
1.1.1 Epidemiology
Breast cancer is the most common diagnosed cancer in the UK despite the fact
that it is rare in men. In 2008, 48,034 new cases of breast cancer were diagnosed in
the UK: over 99 % in women and less than 1 % in men. Moreover, in the last ten
years female breast cancer incidence rates have increased by 6 % in the UK (Cancer
Research UK. Breast Cancer. 2009). The UK breast cancer screening program was
set up in 1988 and uses mammography to screen all woman aged between 50 and
70 every 3 years (Cancer Research UK. Breast Cancer. 2009). However, the majority
of breast cancers are self-detected.
Survival rates for breast cancer vary greatly depending on the breast cancer
stage. Overall, women’s survival rate after five-year is 82 %, 72 % at ten years and
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64 % at twenty years. Despite improvement of the survival rate in the past years,
12,116 deaths from breast cancer were reported in the UK in 2008 and breast cancer
remains the most common cause of death in younger women aged 35-54 years (Fig.
1.1) (Cancer Research UK. Breast Cancer. 2009). Breast cancer accounts for around
16% of female deaths from cancer in the UK annually and causes of death from
breast cancer vary greatly according to the age at diagnosis, node involvement,
cardiovascular condition and osteoporosis. It was found that non-breast cancer-
related deaths were more common than breast cancer-related deaths in a study
involving 50 % of patients younger than 70 years and 50 % of patient over 70 years
old. Two factors have different correlations with the type of death (Chapman, Meng et
al. 2008). Cardiovascular disease was associated with significant increased risk of
death from other causes than breast cancer, and osteoporosis was associated with
significant risk of death from other malignancies. Breast cancer-related death was
associated with lymph node involvement and metastases to other organs (Chapman,
Meng et al. 2008). Between 1989 and 2008 the breast cancer mortality rate fell in
each age range. The reduction in breast cancer mortality rates is likely to be due to
improvements in screenings, specialization of care and the widespread adoption of
tamoxifen treatment since 1992.
Figure 1.1 Number of deaths and ages-specific mortality rates of breast cancer, UK, 2008. Cancer Research UK.
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1.1.2 Breast cancer development
Breast cancer development involves a progression through different mammary
lesions and clinical stages starting from ductal hyperplasia, with subsequent evolution
to atypical hyperplasia, in situ carcinomas progressing into invasive carcinomas (Fig.
1.2) (Allred, Mohsin et al. 2001). Among invasive carcinomas, 80 to 90 % are formed
in breast ducts (referred as ductal carcinoma in situ or DCIS), while 10-20 % occur in
breast lobules (referred as lobular carcinoma in situ or LCIS) (Fig. 1.2).
Mammography is the first examination undergone by women and is followed by
a biopsy. These tests are used to define the breast cancer stage. Stage 1 is defined
by a tumour no wider than 2 centimetres, but not spreading to the lymph nodes. In
contrast, stage 2 refers to a tumour wider than 2 centimetres, which has no sign of
spread to other organs. Stage 3 involves a tumour of 5 centimetres or less that has
reached the lymph nodes, whereas stage 4 is a stage where the tumour is seen in
lymph nodes and has spread to other parts of the body.
1.2 BREAST CANCER CLINICAL MANAGEMENT
For years, mastectomy has been the treatment of choice for all types of breast
cancer. After surgery, radiation is the first therapy used to kill cells left over from
surgery to reduce recurrence rates (Schoenfeld and Harris 2011). Chemotherapies
were introduced in the 1940’s to reduce tumour size before surgery, prevent
recurrence, and treat cancers that have metastasized (Smith and Chua 2006). The
discovery of the anti-oestrogen tamoxifen in 1966 and the identification of distinctive
oestrogen receptor alpha (ERα) and progesterone receptor (PR) status in mammary
tumour cells determined the significance of the hormone receptor status for breast
cancer clinical management. Two types of tumours were established based on ERα
/PR status: those with ERα (generally PR positive) benefit from anti-oestrogen or
endocrine therapy, while those without ERα (generally negative for PR) do not
respond to endocrine therapy. A transmembrane protein, human epidermal growth
factor receptor 2 (HER2), was later found overexpressed on the surface of human
breast cancer cells. Based on the poor prognosis of those patients ERα-/PR-/HER2+
receptor status, a monoclonal antibody specifically targeting the HER2, trastuzumab,
20
was developed and approved for the treatment of breast cancer in 1998 (Hynes and
Stern 1994).
Current clinical management of breast cancer involves patients’ first undergoing
surgery to totally or partially remove the tumour depending on the breast cancer
stage, followed by adjuvant radiotherapy for partially removed tumours. Then,
chemotherapy and hormonal therapy is administrated according to the patient
receptors status. Chemotherapy alone is administrated to ERα/PR/HER2 negative
receptors. ERα/PR positive patients are administrated endocrine therapy in addition
to chemotherapy, while HER2 positive patients will receive HER2-targeted therapy
with chemotherapy (Fig. 1.2) (Smith and Isaacs).
21
Figure 1.2 Two main types of breast cancer and therapies. After surgery, the hormone receptor status is determined. Patients ER/PR/HER2 negative receptors will be administrated chemotherapies, while ER/PR/HER2 positive receptors patients will be treated hormonal therapies in addition to chemotherapies.
22
1.2.1 Breast cancer chemotherapies
1.2.1.1 DNA damage agents
About 10 to 20 % of breast cancer patients are triple negative receptors for
ER/PR/HER2. These patients have poorer prognosis than ERα positive breast
cancer patients because they are only sensitive to chemotherapy agents. So far, no
biomarker has been identified to allow for the development of targeted therapy for
these patients. Chemotherapy agents for breast cancer can be divided into five
categories according to their mechanisms of action: alkylating, anti-metabolite,
topoisomerase inhibitor, anthracycline and anti-mitotic agents (Hortobagyi 1995,
Rodler, Korde et al. 2010, Rodríguez-Lescure 2010).
Alkylating agents are one of the earliest used chemotherapy agents for the
treatment of cancer, which alkylate many nucleophile functional groups in
cells. Cisplatin and carboplatin, as well as oxaliplatin, are alkylating agents and act
directly on DNA, causing cross-linking of DNA strands, abnormal base pairing, or
DNA strand breaks, preventing cells from dividing (Euhus 2011). Alkylating
chemotherapy drugs are effective during all phases of the cell cycle.
The structure of anti-metabolites is similar to those of vitamins, amino acids and
precursors of DNA and RNA, and act by inhibiting cell division. Anti-
metabolites block and prevent purines and pyrimidines from incorporating into DNA
during the synthesis phase (S phase) of the cell cycle, stopping normal development
and division. These anti-metabolites also effectively block RNA synthesis. Examples
of anti-metabolites include 5-fluorouracil (5-FU), methotrexate and gemcitabine.
Anthracyclines are anti-tumour antibiotics that interfere with enzymes involved
in DNA replication (Morris, Hudis et al. 2011). These antibiotics work in all phases of
the cell cycle and are widely used for a variety of cancers. A major dose-limiting
factor when giving anthracyclines is that they can permanently damage the heart if
given in high doses. Examples of anthracyclines include daunorubicin, doxorubicin
(Adriamycin®) and epirubicin.
23
Topoisomerase inhibitors interfere with two enzymes called topoisomerase I
and II, which help separate the strands of DNA during transcription and cell division.
Examples of topoisomerase I inhibitors include topotecan and irinotecan (CPT-11)
while examples of topoisomerase II inhibitors include etoposide (VP-16) and
teniposide.
Mitotic inhibitors are often plant alkaloids and other compounds derived from
natural products. They act to stop mitosis or inhibit enzymes from producing proteins
needed for cell division. They are effective during the mitotic phase (M phase) of the
cell cycle, but can damage cells in all phases. These drugs are known for their
potential to cause peripheral nerve damage, which can be a dose-limiting side effect.
Examples of mitotic inhibitors include taxanes (paclitaxel (Taxol®) and docetaxel
(Taxotere®) (Fig. 1.3).
Clinically, combinations of up to three chemotherapy drugs are administrated
together to achieve maximum efficiency against tumour growth. Some of the most
common combinations used for breast cancer are CMF (cyclophosphamide,
methotrexate and fluorouracil), FEC (epirubicin, cyclophosphamide and fluorouracil),
E-CMF (epirubicin, followed by CMF), AC (doxorubicin (adriamycin) and
cyclophosphamide) (Fig. 1.2) (Chu and Kiel 1982, Sledge, Neuberg et al. 2003,
Blohmer, Schmid et al. 2010, Seidman, Brufsky et al. 2011). The oncologist may offer
a choice of chemotherapy combinations as different combinations have different side
effects.
24
Figure 1.3 Targets for chemotherapies. Alkylating agents cross-link DNA strands and induce DNA strand breaks. Anti-metabolites prevent the incorporation of purines and pyrimidines into DNA in S phase. Anthracyclines inhibit multiple enzymes involved in DNA replication. Topoisomerase inhibitors inhibit topoisomerase enzymes that are required for DNA replication. Mitotic inhibitors inhibit enzymes required for mitotic execution.
25
1.2.1.2 DNA damage response pathways
Taken together, each category of chemotherapies affects DNA directly via
cross-linking, synthesis inhibition, or indirectly via inhibition of enzymes required for
DNA replication. Chemotherapy agents damage DNA and can further induce multiple
DNA damage responses including cell cycle checkpoints, transcriptional activation
and DNA repair.
Cell cycle checkpoints are regulatory pathways governing the order and timing
of cell cycle transitions to ensure accurate completion of the cell cycle phases. The
key regulators of the checkpoint pathways in the mammalian DNA damage response
are ATM (ataxia telangiectasia, mutated) and ATR (ATM and Rad3-related) protein
kinases. Both of these proteins belong to a structurally unique family of serine-
threonine kinases. Although ATM and ATR share similar cellular substrates, they
generally respond to distinct types of DNA damage (Traven and Heierhorst 2005).
ATM is the primary mediator to DNA double strand breaks (DSBs) that can arise by
exposure to ionizing radiation (IR), whereas ATR plays mainly a role in response to
ultraviolet (UV) damage and stalls in DNA replication (single DNA breaks) (Abraham
2001). The key trigger in the G1 cell cycle checkpoint is the activation of p53, which
is phosphorylated by checkpoint kinase 2 (CHK2), downstream target of ATM.
Activated p53 upregulates a number of target genes involved in DNA damage
response (MDM2, GADD45α) and G1 cell cycle arrest (p21Cip1) (Fig. 1.4) (Bartek and
Lukas 2001). During the S phase checkpoint, ATM activates two parallel pathways.
Firstly, ATM phosphorylates CHK2 to prevent cyclin-dependent-kinase 2
(cdk2)/cyclins activation and completion of DNA synthesis. Secondly, ATM
phosphorylates a series of downstream substrates involved in DNA repair including
Nijmegen breakage syndrome (NBS1), breast cancer susceptibility gene 1 (BRCA1)
and structural maintenance of chromosome protein 1 (SMC1). The blockage of entry
into mitosis is essential for the G2 checkpoint activation. ATM and ATR modulate the
phosphorylation status of the cyclin-dependent CDC2 to keep it in its inactive form
and prevent the entry into mitosis (Fig. 1.4) (Zhou and Elledge 2000).
26
Figure 1.4 Cell cycle checkpoints following DNA damage. ATM/ATR activated by DNA damage trigger signalling cascades leading to cell cycle arrest and delay. ATM/ATR induce a series of phosphorylation affecting p53 and MDM2 leading to G1 arrest. ATM/ATR also phosphorylate and stabilize CHK1/2 and the complex NBS1/BRCA1/SMC1, which cause a delay in S phase. G2 arrest is induced by the phosphorylation of CDC25C, which prevent phosphorylation and activation of the CDC2/CYCLIN B1 complex (From R&D systems website).
27
Multiple DNA repair pathways can be activated following DNA alterations.
Chemotherapy agents induce DSBs that are the worst form of DNA damage. The
signal transduction activated by DSBs is strictly dependent on the ATM/ATR family of
serine-threonine kinases that regulates two main DSB repair pathways: the
homologous recombination (HR) and non-homologous end-joining (NHEJ). Emerging
studies have demonstrated there is a cross-talk between the signal of ATM/ATR, and
HR and NHEJ repair mechanisms (Kühne, Tjörnhammar et al. 2003). NHEJ involves
the ligation of two DNA double strand break without sequence homology between the
two DNA ends. Whereas in HR, the damaged DNA retrieves genetic information from
an undamaged DNA that shares sequence homology (San Filippo, Sung et al. 2008).
NHEJ is the dominant repair mechanism in cells undergoing G0, G1 and early S
phases. The central event involves the KU70/80 heterodimer binding to the two ends
of the DSB enabling the recruitment of DNA-dependent protein kinase (DNA-PK).
DNA-PK binds to a single strand of the DSBs, which triggers its kinase activity. One
DNA-PK substrates is X-ray repair cross-complementing protein 4 (XRCC4) that
forms a complex with the DNA ligase IV and stimulates DNA end-ligation (Fig. 1.5).
Prior to the DNA end-ligation, the complex MRE11/RAD50/NBS1 exerts its
exonuclease activity to clean up the DNA ends (Jackson 2002).
HR, particularly important during S and G2 phases, involves DNA ends
resection by nucleases, homology DNA pairs allowing strand invasion, homologous
recombination and DNA repair synthesis. This process can be divided into four steps:
resection, strand invasion, branch migration and Holliday junction formation. The
nucleolytic resection of the DNA in the 5’ to 3’ is an early event involving the complex
MRE11/RAD50/NBS1 (Fig. 1.5). The resulting 3’ single strand DNA ends are then
bound by RAD51, which is a process involving the binding replication protein A
(RPA), RAD52, RAD54 and BRCA1, BRCA2. The RAD51 nucleoprotein filament then
interacts with the homologous undamaged DNA and catalyses strand exchanges, in
which the damaged DNA invades the undamaged DNA. The 3’ terminus of the
damaged DNA is extended by a DNA polymerase, which copies genetic information
from the undamaged DNA molecule. Finally, the ends are ligated using DNA ligase I
and the DNA cross-overs, called Holliday junctions, are cleaved and ligated to two
intact DNA molecules (Jackson 2002, Mao, Bozzella et al. 2008).
28
Figure 1.5 Double strand break repair. Homologous recombination process uses a homologous chromosome to repair DNA. When no homologue is present, breaks can be repaired with the non-homologous end-joining mechanism, in which two ends are ligated.
29
1.2.2 Breast cancer endocrine therapies
1.2.2.1 Anti-oestrogen therapies
About 70 to 80 % of breast cancer patients are ERα positive (in which more
than half are also PR positive) and ERα positivity predicts for response to endocrine
therapy (Kuukasjärvi, Kononen et al. 1996, Bauer, Brown et al. 2007). Therefore,
anti-oestrogen therapies are usually given to patient diagnosed ERα positive when
surgery and neoadjuvant chemotherapy were already administrated.
Tamoxifen (OHT) has been the standard adjuvant anti-oestrogen therapy for
pre- and post-menopausal women with ERα and/or PR positive breast cancer for
years. It is a selective ERα modulator (SERM) that functions as ERα antagonist in
breast and as ERα agonist in the heart and bones (Smith and Chua 2006).
It blocks the binding of oestrogen and consequently its activity on ERα.
Tamoxifen binding further prevents critical ERα conformational changes that are
required for the association of co-factors and the transcriptional activity of ERα.
However, the oestrogen-agonist effects of tamoxifen have been associated with
serious life-threatening events including endometrial cancer, stroke and trombo-
embolism. Selective ERα down-regulators (SERD) such as fulvestrant (ICI182,780)
are used as alternatives to tamoxifen (Krell, Januszewski et al. 2011). In contrast to
tamoxifen, SERDs are pure antagonists that bind with 100 fold greater affinity,
inhibiting receptor dimerization and abrogating oestrogen signalling. Clinical studies
have also shown that fulvestrant decreases ERα expression levels (Dauvois et al.
1992). Third-generation aromatase inhibitors (AIs) have also been introduced as an
alternative to tamoxifen therapy for post-menopausal women which cancer has
progressed following tamoxifen treatment. AIs prevent oestrogen synthesis by
inhibiting the action of aromatase, an enzyme necessary for the conversion of
androgens to oestrogens (Fig. 1.6A).
30
Figure 1.6 Effects of anti-oestrogens. A. ERs are free in the cytoplasm and can be bound by oestrogen to translocate to the nucleus, and recruit ER co-activators to activate ER-responsive genes transcription. When the anti-oestrogen OHT is added, it binds to ER, translocate to the nucleus and recruit ER co-repressors which inhibit gene transcription. In contrast, ICI binds ER and guides it to the proteasome for degradation. B. The cell cycle is divided in 4 phases which are regulated by different cyclin/cdk complexes. When cells are treated anti-oestrogens, cells arrest in G1 or G2 phases through the downregulation (-, in pink) of cyclin D1 and E and the upregulation (+, in blue) of cdk inhibitors and hypophosphorylated Rb. Adapted from Dehay and Kennedy, 2004.
31
The effect of anti-oestrogen therapies has been studied since 1975 and shown
to induce cell cycle arrest in G1 resulting in a lower proportion of cells in S phase.
Studies in MCF-7 breast carcinoma cells revealed a decrease of CYCLIN D1 mRNA
level and suggested that cyclins involved in G1 phase might be the target for anti-
oestrogens to block cells entry in S phase (Lykkesfeldt, Madsen et al. 1994). Further
studies using the pure ERα antagonist, ICI182,780, demonstrated a decrease in S
phase cells combined with increased of hypophosphorylated Retinoblastoma protein
(RB), which inhibits the transcriptional activity of E2F restricting the transcription of
cyclins and cdks. Additionally, treatment of MCF-7 cells with the pure ERα antagonist
decreased CYCLIN D1 at mRNA and protein levels. Studies of cyclin-dependent
kinases revealed no change in their expression pattern. Anti-oestrogens treatment of
breast cancer cell lines also showed an upregulation of two members of the cdk
inhibitors Cip1/Kip1 family, p27Kip1 and p21Cip1, which by consequent inhibit G1 and
G2 cyclins (Fig. 1.6B). Tamoxifen can also induce apoptosis at a higher
concentration of 5 µmol/L in MCF-7 cells. Protein kinase C, C-MYC and p53 were
identified as potential targets in triggering tamoxifen-induced apoptosis but the exact
mechanism still remains elusive (Doisneau-Sixou, Sergio et al. 2003).
1.2.2.2 The oestrogen receptor pathway
Oestrogens, 17ß-estradiol (E2), estrone and estriol, are the most important
regulators of breast cancer growth. Oestrogens are steroid hormones produced
primarily by the developing follicles in the ovaries and the placenta, and act through
the oestrogen receptors, ERα and ERß, products of different genes.
The oestrogen receptors belong to the nuclear receptor superfamily of
transcription factors, which includes steroid hormones, thyroid hormones, vitamin D
and retinoic acid. ERα and ERβ are composed of three functional domains: the N-
terminal, DNA binding domain (DBD), and the ligand-binding domain (LBD) (Fig. 1.7)
(Ruff, Gangloff et al. 2000). The N-terminal domain of nuclear receptor encodes a
ligand-independent activation function (AF-1) that is involved in protein-protein
interactions, and transcriptional activation of target gene expression. The DNA
binding domain contains a two zinc finger structure, which plays an important role in
32
receptor dimerization and in binding within the oestrogen response element (ERE).
The LBD mediates ligand binding, receptor dimerization, nuclear translocation, and
transactivation of target gene expression. The activation function 2 (AF-2) in the LDB
is governed by the binding of ligands (Hall and McDonnell 2005).
Figure 1.7 Direct interactions of oestrogen receptor with its cofactors. Oestrogen (E2)-bound oestrogen receptor (ER) directly interacts with co-activators SRC-1, GRIP1 or AIB-1 at the AF-1 or AF-2 domain (shown by green lines). Tamoxifen (OHT)-bound ER recruits co-repressors NCOR and SMRT to the AF-2 domain (shown by red line).
Oestrogen receptors act as transcription factors, either by activating or inhibiting
the expression of a wide array of genes. ERα and ERß share a high degree of
homology, 97 % in the DBD and 60 % in the LBD, and therefore interacts with the
same oestrogen ligand (Bai and Gust 2009). However, the expression and
characteristics of these receptors differ. ERα is widely expressed and is predominant
in the breast, uterus and bone, while ERß is mostly expressed in ovary, prostate,
testis, lung, thymus, spleen and areas of the brain. Animal models have shown that
ERß can function as an inhibitor of ERα, and is often downregulated in breast
tumours. While ERα activity induces breast cancer cell proliferation and
tumourigenesis in mice under estradiol, ERß is anti-proliferative and prevents the
formation of tumours (Paruthiyil, Parmar et al. 2004).
The ERs are sequestered in a multi-protein complex, including heat shock
proteins, in the cytoplasm of cells until their activation by ligands (Fig. 1.7). The
binding of the ligand on ER induces its conformational changes, promoting its
dimerization, its binding to DNA and the recruitment of co-factors inducing
transcription of target genes. Among ER co-factors, some ER modulators bind to the
33
AF-2, including ER-interacting proteins 140 and 160 (ERAP140 and ERAP160),
receptor interacting proteins 140 and 160 (RIP140 and RIP160) and the p160 family
(SRC-1, GRIP1, AIB1) (Halachmi, Marden et al. 1994, Oñate, Tsai et al. 1995). In
addition to co-factors that modulate ER activity by binding to AF-2, AF-1 interacting
co-factors have also been described, such as p68 RNA helicase, which enhances ER
activity through AF-1 (Endoh, Maruyama et al. 1999). While co-activators enhance
ER activity, co-repressors decrease the agonist effect of oestrogens. The first
identified and most studied are the nuclear receptor co-repressor (NcoR) and
silencing mediator for retinoid and thyroid hormone receptors (SMRT). Cloning of
cDNAs stimulated by oestrogen resulted in the identification of numerous target
genes including pS2 and CATHEPSIN D, CYCLIN D1, C-MYC and many others
(Elangovan and Moulton 1980, Brown, Jeltsch et al. 1984, Dubik and Shiu 1988,
Altucci, Addeo et al. 1996). Promoter region analysis of these genes led to the
discovery of two distinct mechanisms of ER binding, the “classical” and “non-
classical” binding as genomic response (Fig. 1.7). The former involves the binding of
ER within the ERE, while the latter involves the binding of DNA through a different
motif including those for AP-1 and Sp-1 (Klinge 2001, Carroll, Meyer et al. 2006).
Another mechanism, by which ER regulates transcription indirectly, referred to as the
non-genomic response, identified about sixty years ago. Oestrogen binding sites
were identified on the membrane of endothelial cells. Further studies showed that ER
can mediate rapid signals originating from the membrane into the nucleus by the
activation of growth factor receptors, tyrosine kinases, mitogen-activated protein
kinase (MAPK) and phosphatidylinositol 3 kinases (PI3K) (Fig. 1.7) (Migliaccio, Di
Domenico et al. 1996, Migliaccio, Piccolo et al. 1998, Simoncini, Hafezi-Moghadam
et al. 2000, Song, McPherson et al. 2002).
The success of targeted anti-oestrogen therapy motivated the search for
genetic mutations occurring in breast cancer and the development of new targeted
therapies.
34
1.3 GENETIC AND NEW THERAPIES
1.3.1 Genetic predispositions and mutations
Risk factors for breast cancer can be divided into environmental and genetic
categories. A number of environmental risks related to reproductive events include
age, menstruation age, age at first birth, menopause and exogenous uptake. In
addition, a strong family history involving genetic alterations is recognized as the
strongest risk factor.
Genetic predisposition to cancer occurs in a minority of patients and is
conferred by inherited mutations in high penetrance genes. The BRCA1 and BRCA2
genes have been identified in the 1990’s as “high risk” breast cancer susceptibility
genes. Women carrying deleterious mutation of these genes have 45-65 % risks of
developing breast cancer (Ahmed, Lalloo et al. 2009). Women with very strong family
history can be tested for faulty BRCA genes, but account for just 3 %.
While some of these alterations might be inherited, most accumulate during a
woman’s lifetime. Gene alterations accumulated with time are known as somatic
mutations that are permanent and transmissible in genetic material. A wide variety of
genes is commonly mutated or incorrectly regulated in breast cancer cells and have
been implicated in the development and progression of the disease. These genes
encompass growth factors receptors and their ligands, intracellular signalling
molecules, cell cycle regulators, apoptosis regulators and adhesion molecules
leading to cell proliferation, inhibition of apoptosis and invasion (Cordon-Cardo 1995,
She, Chandarlapaty et al. 2008). Studies of these altered molecules are promising as
new potential targets for breast cancer therapy. The best example of such a therapy
is trastuzumab, which has been shown to be effective in breast cancers
overexpressing the growth factor HER2 (Bartsch, Wenzel et al. 2007).
35
1.3.2 Targeted therapies
The human epidermal growth factor receptor/HER family of transmembrane
type I receptor tyrosine kinases are enzymes that are fundamental in cellular
processes such as proliferation, differentiation and survival. The members of this
family contain an extracellular ligand-binding domain, a single membrane-spanning
region, a nuclear localization signal, and a cytoplasmic tyrosine kinase domain.
HER1, HER3 and HER4 members interact with specific ligands, while no natural
ligand has been identified for HER2. HER2 can be activated by hetero-dimerization
with ligand-activated HER co-receptors, which modulate receptor internalization and
prolong signal transduction (Prenzel, Fischer et al. 2001, Li and Hristova 2010).
Conformational changes occurring of dimer receptors lead to auto-phosphorylation
and initiation of several signal transduction cascades. These type I receptors transmit
signals through the Ras/Raf/mitogen-activated protein kinase/extracellular signal-
regulated kinase (ERK) pathway stimulating cell division (Fig. 1.8). Cell lines studies
suggest that these receptors modulate cell survival via the activation of AKT/PI3K
pathway. In vitro and in vivo studies have established oncogenic properties (Hubalek,
Brunner et al. 2010). Aberrant HER1 and HER2 signalling have been associated with
cancer cell proliferation and survival. Activated HER2 was first identified by a point
mutation in rat neuroblastomas (Perantoni, Rice et al. 1987). It was later found to be
overexpressed in 25 % of some human breast cancers. The prognosis of those
patients with HER2 expression on breast cancer cells surface is poor, which
warranted the development of antibodies and small molecules specifically targeting
this receptor.
36
Figure 1.8 HER2 signalling and pathway targeted therapy. Hetero-dimerization of HER2, induced by the ligands of HER members, activates the phosphoinositol 3-kinase pathway including AKT leading to cell survival and the mitogen-activated protein kinases promoting cell proliferation (Hubalek et al 2010).
37
In the 1980’s, the monoclonal antibody against HER2, trastuzumab, was
developed and approved in 1998 for the treatment of metastatic breast cancer. In
2005, five adjuvant trials evaluated trastuzumab as a potent agent for early breast
cancer with higher benefit than adjuvant chemotherapy and similar to that seen with
adjuvant endocrine therapy (Harries and Smith 2002). Although trastuzumab’s
method of inhibiting HER2 activity is not fully understood, studies suggest that the
drug promotes internalization and degradation of HER2, or disrupts the activation of
the AKT/PI3K pathway. Trastuzumab administration leads to cell cycle arrest at the
G1/S boundary, which often results from an increase of p27Kip1 level, and a
subsequent decrease of CYCLIN D1 and cdk2 (Baselga, Albanell et al. 2001).
Currently, trastuzumab is the only HER2-targeted therapy approved by the FDA for
the treatment of early and metastatic breast cancer overexpressing HER2, therefore
evaluating HER2 expression has become mandatory in early breast cancer (Fig. 1.8).
1.4 BREAST CANCER RECURRENCE
1.4.1 Recurrence of the disease
The recurrence rate among patients who did not receive adjuvant endocrine
therapy is nearly 50 % throughout the first 10 years after diagnosis.
There are so many factors that account into the risk of recurrence. Some
common indicators of recurrence include the involvements of lymph node and
vasculature, the tumour size, the histological grade, the receptor status, proliferative
capacity, oncogene expression and somatic mutation (Smith and Chua 2006).
Recurrent breast cancer can be categorized in three types: local recurrence
appearing at the tumour site which may be considered as a failure of initial treatment,
regional recurrence showing that cancer has spread is very common and distant
recurrence or metastasis which is the most serious type and is associated with
decreased patient survival. In 65-75 % of distant recurrences the breast cancer then
spreads from the lymph nodes to the bones. In rare cases, breast cancer may also
metastasize to other sites including the lungs, liver, brain or other organs.
38
1.4.2 Chemotherapy resistance
Resistance to chemotherapies is a major problem in the clinical management of
breast cancer and most particularly for triple negative patients, who can only be
administered chemotherapy drugs. Response rate for first line chemotherapies is
between 30 and 70 %. However, these agents are efficient only for 6 to 10 months.
Patients who initially respond become resistant to the initial agents and to multiple
anti-cancer drugs, which have different structure and mechanism of action. This
phenomenon is referred as multidrug resistance (MDR).
Chemotherapy resistance can be mediated by various mechanisms such as an
increased activity of exporters or decreased activity of importers resulting in reduced
intracellular drug concentrations or reduction of cellular uptake, respectively. Drug
transport is mediated by the ATP-binding cassette transporters (ABC-transporters)
(Coley 2008). An increased of the ABC-transporter P-glycoprotein (PGP) expression
from 11 % to 30 % in patients, who had received doxorubicin and taxol, correlated
with drug resistance (Leonessa and Clarke 2003). Furthermore, multidrug resistance-
associated protein 1 (MRP1) is frequently found to be overexpressed in primary
breast cancer and associated with relapse of node-positive and –negative patients
who received cyclophosphoamide, methotrexate and 5-fluorouracil (Riordan and Ling
1985, Tsuruo 1988). Additionally, the activation of detoxifying systems also allows
cells to escape to the effect of chemotherapies. Upregulation of the cytochrome
P450, aldehyde dehydrogenase and glutathione S-transferases particularly affect the
toxicity of cyclophosphamide. Resistance can also be the consequence of defective
apoptotic pathways or a change in cell cycle checkpoint. P53 is lost in cells where the
cytotoxic effect of doxorubicin is delayed or lost. Mutations in p53 have been linked to
doxorubicin resistance and to early relapse in breast cancer patients. Comparisons
between mouse fibroblast cells containing wild-type p53 and p53 knock-out showed
that the absence of p53 reduces apoptotic cell death and induces doxorubicin
resistance. Mutated p53 loss of function was also associated with the abolition of
p21Cip1 transcriptional activation resulting in cell cycle arrest defect. Expression of the
anti-apoptotic family of protein BCL-2 has been detected in 80 % of breast cancers
from women with primary tumours and having either node positivity or negativity. In
contrast, the expression of the pro-apoptotic factor BAX is lost in some breast
39
tumours (Krajewski, Krajewska et al. 1999). Reduced BAX levels correlate with
shorter overall survival, fast tumour progression and failure to respond to therapy
(Coley 2008).
1.4.3 Anti-oestrogen therapy resistance
Currently, tamoxifen remains the treatment of choice for most women with ERα
positive invasive breast carcinomas. Approximately 30 % of ERα positive breast
cancer patients become resistant to tamoxifen. While anti-oestrogens have been
available since the early 1970’s, the mechanisms of action and development of
resistance is still not fully understood. There are two forms of anti-oestrogen
resistance: de novo resistance and acquired resistance. By definition, de novo
resistance is present before drug exposure, while acquired resistance occurs after
drug exposure. Additionally, endocrine resistance can be categorized into loss or
mutation of ERs, specific resistance to anti-oestrogens, modified ERα interaction
proteins and ligand independent ERα activation (Clarke, Liu et al. 2003).
Since the effects of anti-oestrogens are mediated through ERs, the degree of
ER expression is a strong predictor of response. Loss of ER expression is the
primary mechanism of de novo resistance to tamoxifen with ER/PR negative.
However, the lost of ER occurs only in 15 % of cases (Ring and Dowsett 2004). In
addition to genetic modifications, epigenetic changes such as CpG island
hypermethylation causes transcriptional inactivation of ER gene (Issa, Ottaviano et
al. 1994). In a study, the analysis of the methylation status of CpG dinucleotides in
ER from patients with recurrent breast cancer following tamoxifen allowed the
development of a predictive score that could be used to identify patients likely to
respond to tamoxifen (Martens, Nimmrich et al. 2005). Further studies are
undertaken to confirm the validity of this approach. A second ER was cloned from a
rat prostate cDNA library and was named ERß (Kuiper, Enmark et al. 1996). ERß is
highly homologous to ERα and binds to the same ligands but has different effects on
gene transcription. There is divergent data concerning the expression of ERß, patient
prognosis and anti-oestrogen responsiveness (Omoto, Inoue et al. 2001, Fuqua,
Schiff et al. 2003, Iwase, Zhang et al. 2003). However, evidence suggest that ERß
40
protein expression is decreased in breast carcinoma compared to normal or benign
lesions (Roger, Sahla et al. 2001).
The majority of patients that initially respond to tamoxifen acquire resistance
after long-term exposure without losing ERα expression. Therefore, most of the
published information on potential mechanisms of resistance has been documented
by studies in ERα positive breast cancer cells selected by sustained exposure to anti-
oestrogen.
A mechanism of drug resistance common to chemotherapies and endocrine
therapies is the emergence of increased drug efflux or reduced drug influx. Intra-
tumoural tamoxifen concentration was decreased in tamoxifen-resistant breast
cancer compared to tamoxifen-responsive breast cancer. Although the mechanism
responsible for altered tamoxifen accumulation is not understood, in vitro study
shows that overexpression of MDR1 in MCF-7 cells reduces tamoxifen sensitivity
(Clarke, Currier et al. 1992). In addition, lower concentration of tamoxifen active
metabolite (4-hydroxy-N-desmethyltamoxifen) was found in patients carrying a
variant of the CYP2D6 allele (Stearns, Johnson et al. 2003).
ERα is activated by binding of estradiol that induces recruitment of co-factors,
conformational changes, phosphorylation of ERα, and its dimerization before binding
of the ERE within the promoter of ER-responsive genes. This is referred to as the
classical mode of action. The overexpression of AIB1, an ER co-activator, is
observed in 50 % of breast tumours (Anzick, Kononen et al. 1997). AIB1 is also
highly expressed in MCF-7 breast cancer cell line and in a mouse xenograft (List,
Lauritsen et al. 2001). The overexpression and phosphorylation of AIB1 led to
constitutive transcriptional activity of ERα conferring resistance in vitro and in
xenograft models (Musgrove and Sutherland 2009). In addition, AIB1 overexpression
is associated with reduced responsiveness to treatment in breast cancer patients and
a worse disease-free survival (Osborne, Bardou et al. 2003, Alkner, Bendahl et al.
2010). In vitro studies suggest that overexpression of other co-activators such as
SCR-1, may also be able to enhance ER activation by oestrogen and agonist activity
of tamoxifen. However, no clinical data have verified SCR-1 role in tamoxifen
resistant patients. While ER co-activators levels are found overexpressed in tumours
that acquired tamoxifen resistance, NCoR levels, an ER co-repressors, are declined
(Chan, Lykkesfeldt et al. 1999).
41
ER can also regulate gene expression by interacting with DNA directly via other
transcription factors, such as FOS/JUN activating protein 1 (AP-1) and NF-κB (non
classical mode) (Nilsson, Mäkelä et al. 2001). The increase in transcriptional activity
elicits an increase in ERα activity, which is associated with tamoxifen resistance.
Post-translational modifications, such as phosphorylation, acetylation and
methylation, affecting ERα can also influence its interaction with factors and lead to
anti-oestrogen insensitivity. Numerous studies showed cross-talk between ERα and
growth factor receptor pathways, such as HER family and insulin-like growth factor
(IGFR) family. ERα can be phosphorylated on several sites but the most reported is
serine 118. ER can be phosphorylated at serine 118 by the MAPK ERK1/2, which is
downstream of HER2 signalling pathway (Kato, Endoh et al. 1995). Phosphorylation
enhances ERα ligand sensitivity and may lead to ligand-independent activation.
Indeed, ERK1/2 expression and activity are increased in endocrine resistant breast
cancer cell lines (Kronblad, Hedenfalk et al. 2005). Upstream RAS/MAPK pathway
can be activated by IGF stimulation inducing phosphorylation of ERα serine 118 and
resulting in enhanced activation (Kato, Endoh et al. 1995). A direct interaction
between ERα and IGFR leads to activation of IGFR downstream targets, thereby
leading to an increase in cell survival (Ring and Dowsett 2004). In MCF-7 breast
cancer cell line, phosphorylation of ERα on serine 118 and serine 167 by the receptor
tyrosine kinase RET and mTOR pathway leads to ligand-independent activation of
ER-responsive genes and tamoxifen resistance (Morandi, Plaza-Menacho et al.
2011). The clinical relevance of serine 118 phosphorylation has not been yet
established; some studies showed a bad prognosis correlated with phospho-serine
118, while other studies positively correlated phospho-serine 118 with response to
endocrine treatments (Sarwar, Kim et al. 2006, Riggins, Schrecengost et al. 2007,
Yamashita, Nishio et al. 2008).
1.4.4 Targeted therapy resistance
The anti-tumour effects exerted by the anti-HER2 antibody, trastuzumab,
require modulation of key signalling pathways and cell cycle/apoptosis regulatory
proteins that are not directly regulated by trastuzumab itself. Therefore, alterations in
these pathways and regulatory proteins limit the therapeutic efficacy of trastuzumab
42
leading to resistance. Studies have shown that high expression of other HER family
members or other growth receptors lead to trastuzumab resistance. For example,
IGFR overexpression activates the AKT/PI3K pathway and renders trastuzumab-
sensitive HER2-overexpressing SKBR3 cells resistant to treatment (Lu, Zi et al.
2001). The expression level of a receptor tyrosine kinase MET was increased in
HER2-overexpressing breast cancer tumour samples with resistance to several
targeted therapies (Shattuck, Miller et al. 2008). Aberrant regulation of the
downstream signalling pathway of HER family, such as phosphoinositol 3-kinase
pathway, also leads to resistance (Campbell, Russell et al. 2004). Furthermore,
trastuzumab can induce the release of HER ligands conferring resistance to its anti-
proliferative effect (Kong, Calleja et al. 2008). In addition to the high levels of ligands,
the membrane-associated glycoprotein mucin-4 (MUC4), which is overexpressed in
breast cancers, can mask HER2 interfering with trastuzumab binding (Nahta and
Esteva 2006). Several studies also identified a truncated version of HER2 which
lacks the extracellular domain (p95 HER2) and able to escape trastuzumab´s
binding. In a survey, Molina et al. found HER2 truncation more highly expressed in
surgically excised node-positive breast cancer samples than node-negative ones
(Molina, Sáez et al. 2002). Consistently, the comparison between MCF-7 transfected
with HER2 and p95HER2 showed that only MCF-7/HER2 cells are sensitive to
trastuzumab (Scaltriti, Rojo et al. 2007). The recruitment of phosphoinositol 3-kinase
by a tyrosine kinase receptor catalyses the conversion of membrane-associated
phosphatidylinositol 4,5-biphosphoate (PIP2) to 3,4,5-triphosphate (PIP3). PTEN is
the negative regulator of Class I PI3K converting PIP3 back to PIP2 and has been
reported to be frequently lost (26 %) and mutated (6 %) in breast cancer.
Furthermore, in vitro and in vivo studies demonstrated that knocking-down PTEN in
HER2-overexpressing breast cancer cells induces trastuzumab resistance (Nahta
and Esteva 2006). Additionally, somatic mutations in phosphoinositol 3-kinase
catalytic unit (PIK3CA) were identified in 2004 in several malignancies including
breast cancer (Campbell, Russell et al. 2004). In vitro studies showed that gain-of-
function mutation of the PIK3CA gene lead to increased resistance to trastuzumab in
breast cancer cells than breast cancer cell without PIK3CA mutation. Finally, p27Kip1
expression responsible for the G1/S blockage induced by trastuzumab is reduced in
resistant cells derived from SKBR3 and sensitivity is restored by reintroduction of
p27Kip1 (Chang 2007).
43
1.4.5 Potential strategies overcoming drug resistance
In breast cancer and leukaemia, expression of multidrug pump is observed in
several tissues prior to exposure to chemotherapy and subsequently dramatically
increased once resistance develops. Targeting of the pumps by small-molecular
compounds is an attractive strategy to overcome MDR in cancer. Several inhibitors
of pumps have been developed and are currently under clinical phased studies in
different cancers. To increase the selectivity of MDR inhibition, gene silencing
strategies were developed. Antisense oligonucleotide targeting MDR1 mRNA
partially resensitizes the human MDR xenograft in mice to doxorubicin (Yagüe,
Higgins et al. 2004). Similarly, silencing of MDR1 by RNAi reverses the resistance
of doxorubicin-resistant leukemia cells to doxorubicin and taxol (Wu, Hait et al.
2003). The application of shRNA strategy showed a successful knock-down in vivo
(Yagüe, Higgins et al. 2004).
Since chemotherapy agents exert their anti-tumour effect through production
of DNA damage, inhibition of DNA repair machinery can be used. Poly (ADP-
ribose) polymerase 1 (PARP1) belongs to a large family of nuclear enzyme that are
activated by and recruited to the sites of DNA damages. PARP1 catalyses the
transfer of NAD+ to acceptor proteins and induce the formation of poly (ADP-
ribose) polymers important for the recruitment of the base excision repair
machinery to the sites and repair of DNA. Based on the fact that PARP1 has a role
on DNA repair, inhibition of PARP1 and consequently DNA repair on tumour
resistant to chemotherapies effects may represent a good strategy. Two
therapeutic strategies employ PARP inhibitors in the treatment of cancer. The first
is the use of PARP inhibitors as sensitizers to DNA damaging chemotherapy
agents, while the second aims to exert anti-tumour effect through production of
DNA damage. Indeed, PARP inhibitors enhance cytotoxicity of DNA methylating
agents (Veuger, Curtin et al. 2004). In 2005, pivotal evidence showed that the use
of PARP inhibitors in cells BRCA1 and BRCA2 genes deficient resulted in selective
cytotoxicity compared to wild-type or heterozygous (Bryant, Schultz et al. 2005,
Farmer, McCabe et al. 2005). Based on this, a number of PARP inhibitors are
currently in development for the treatment of cancers including breast cancer. The
BRCA1 and BRCA2 proteins are described for their role in homologous
44
recombination, but BRCA proteins are also implicated in nucleotide excision and
base excision repairs (Hartman and Ford 2002, Alli, Sharma et al. 2009). If PARP
is inhibited, repair-associated breaks result in replication fork-mediated double
strand break, which required BRCA1 and BRCA2-associated recombination
(Arnaudeau, Lundin et al. 2001). Following successes in phase I and II, many
pharmaceutical companies are now examining the efficacy of their PARP inhibitors
in patients BRCA mutation-associated and triple negative breast cancers.
The use of signal transduction inhibitors represents a promising therapeutic
approach as overactivated growth factor signalling pathways are involved in
endocrine resistance of breast cancer. Drugs blocking the signalling pathways of
HER1, IGFR1, MAPK and AKT/PI3K are advanced in clinical development
(Baselga 2011). Results from pre-clinical studies suggest that these drugs can be
effective in both tamoxifen sensitive and resistant breast cancer patients. Indeed,
several reports showed that additive or synergistic effects are obtained in ERα
positive breast cancer patients when treated with a combination of tamoxifen and
signal transduction inhibitors such as tyrosine kinase and farnesyltransferase
inhibitors (Johnston 2005, Baselga 2011). Similar results were obtained with the
combination of mammalian target of rapamycin (mTOR) antagonists and an
aromatase inhibitor letrozole in cell lines models (Baselga 2011). A synergistic
effect has been observed when combining trastuzumab with tamoxifen in ERα
positive and HER2 overexpressing BT-474 breast cancer cell lines (Chen, Wang et
al. 2008). Moreover, treatment of MCF-7 cells with tamoxifen and gefitinib, a HER1
tyrosine kinase inhibitor (TKI), showed greater effects than tamoxifen alone by
inhibition of cell growth and promotion of apoptosis (Gee, Harper et al. 2003). This
pre-clinical data led to the development of clinical trials examining the combination
of tamoxifen and signal transduction inhibitors in ERα positive breast cancer
patients.
Because ERα co-activators are important in ERα function, their
overexpression contributes to endocrine resistance. As mentioned, AIB1 is
amplified in breast cancer and correlate with ERα and PR positive cells. Pre-clinical
studies showed that overexpression of co-activator AIB1 increases tamoxifen
agonist activity suggesting that histone acetyl transferase (HAT) activity is
increased to allow gene transcription. Conversely, ERα co-repressor NCOR1 is
45
underexpressed in tamoxifen resistant mouse models (Lavinsky, Jepsen et al.
1998, Chan, Lykkesfeldt et al. 1999). Therefore, the treatment of breast cancer
cells with histone deacetylase (HDAC) has been developed. TSA and SAHA
treatments, two HDAC inhibitors, reduced breast cancer cell growth. In MCF-7
cells, TSA induces CYCLIN D1 and ERα proteins degradation and derepression of
p21Cip1 resulting in G1 arrest (Butler, Zhou et al. 2002, Alao, Stavropoulou et al.
2006, Kim, Bang et al. 2006, Munster, Thurn et al. 2011).
Currently, trastuzumab is the only HER2-targeted therapy approved by the
FDA for the treatment of metastatic breast cancer overexpressing HER2. However,
the HER2 overexpressing patients who originally responded to trastuzumab
develop resistance. Therefore, the combination of trastuzumab with novel agents
may increase the magnitude and duration of the response. Among novel biological
agents, pertuzumab is a HER2 monoclonal antibody that blocks dimerization of
HER2 with HER1 and HER3, and their signalling pathways. The combination of
trastuzumab and pertuzumab showed synergistic effect inducing apoptosis in
HER2 overexpressing breast cancer cells, but had no effect on trastuzumab
resistant breast cancer cells (Nahta, Hung et al. 2004, Tanner, Kapanen et al.
2004). This result suggests a cross-resistance to HER2 antibodies that are not yet
understood. An alternative is to produce antibody-toxin conjugates. However, the
major limitation of this strategy is the activation of immune response.
In addition to anti-HER2 antibody strategy, TKIs that directly inhibit the
cytoplasmic tyrosine kinase of the growth factor receptor are in development.
Currently clinical trials are being conducted and showed a reduction in HER1 and
HER2 phosphorylation after treatment. Lapatinib, TKIs inhibiting both HER1 and
HER2, is currently being tested in clinical trials (Moy and Goss 2006, Sridhar, Hotte
et al. 2010). Lapatinib has shown remarkable activity in vitro and in vivo including
inhibition of MAPK and AKT activation and growth arrest and apoptosis in HER1- and
HER2-dependent tumours (Xia, Mullin et al. 2002).
To date, much of the information on mechanisms of resistance has come from
cell line studies and too few genes have been considered. The rate, at which breast
cancer relapses, calls for new approaches to provide new target for the development
of treatment capable of reversing drug resistance.
46
1.5 FORKHEAD BOX TRANSCRIPTION FACTORS
A forkhead box transcription factor was discovered in Drosophila in 1989 by
Deftlef Weigel, who identified a novel structure and nuclear localization of a protein
(Weigel, Jürgens et al. 1989, Weigel and Jäckle 1990). The structure described was
three α-helical domains at the N-terminal region, three β-sheets and two large loop
regions located at the C-terminal end, forming a structure which is similar to the
wings of a butterfly, leading to the term winged-helix family. The comparison between
the forkhead box factor and HNF-3A, a gene cloned from a rat hepatocyte, showed
striking similarity (Lai, Prezioso et al. 1990). Fifty-five members grouped into 17
subfamilies (A-Q) (Myatt and Lam 2007) were then identified and described in
vertebrates which have been given a wide range of names until Kaestner et al unified
the nomenclature and the proteins name became FOX (Kaestner, Knochel et al.
2000). The FOX family is an extensive family in which members share greater than
90 % in their winged-helix forkhead DNA-binding domain (DBD) sequences (Clark,
Halay et al. 1993, Kaestner, Knochel et al. 2000). Outside the DBD, FOX proteins
differ significantly leading to differential function and regulation in many processes
including metabolism, proliferation, development and differentiation, aging,
angiogenesis, DNA repair and apoptosis.
1.6 FORKHEAD BOX M1 (FOXM1)
1.6.1 Structure
FOXM1 protein contains three regions: the N-terminal Repressor Domain
(NRD) followed by a conserved DNA Binding Domain called Forkhead winged-helix
domain (FKH). The C-terminal region harbours the transactivation domain (TAD) with
several cyclin/cyclin-dependent-kinase-dependent phosphorylation sites (Fig. 1.9)
(Yao, Sha et al. 1997). FOXM1 can transactivate target genes through two
mechanisms. First, FOXM1 transactivates genes via the binding of its DBD to
FOXM1 binding sites in the promoter of the gene (Wierstra and Alves 2006). Second,
FOXM1 binds human promoters through the binding of FOXM1 DBD to TATA-boxes
47
and the binding of its central domain to TATA-binding protein (TBP) (Wierstra and
Alves 2006). FOXM1 binds to the DNA binding sequence of genes it regulates and
facilitates binding of RNA polymerase to transcribe genes.
Figure 1.9 FOXM1 structure. FoxM1 protein contains 3 main regions: the N-terminal Repressor Domain (NRD). This region is followed by a conserved DNA Binding Domain called Forkhead winged-helix domain (FKH). The C-terminal region harbours the Transactivation Domain (TAD) with several activating Cyclin-Cdk-dependent phosphorylation sites. FoxM1 transcriptional activity also requires the presence of appropriate mitogenic signals involving the Raf/MEK/MAPK signalling pathway which phosphorylate FOXM1 in two sites.
1.6.2 Regulation
A partial fragment of FOXM1, named after WIN, was originally cloned from a rat
insulinoma cell line and detected in human thymus, testis, lung and intestine (Yao et
al. 1997). This fragment WIN was previously isolated in a screen for phospho-
proteins in the mitotic phase, suggesting its regulation by phosphorylation
(Westendorf, Rao et al. 1994, Yao, Sha et al. 1997). Study of FOXM1 in mouse
48
identified that its expression correlated with cycling cells (Korver, Roose et al. 1997).
It is the only forkhead transcription factor known to be associated with proliferation
and that displays a proliferation specific expression pattern (Costa, Kalinichenko et
al. 2003, Laoukili, Stahl et al. 2007). It is expressed in embryos and proliferating
tissues as well as in response to injury in adults (Yao, Sha et al. 1997, Ye, Holterman
et al. 1999, Leung, Lin et al. 2001, Wang, Krupczak-Hollis et al. 2002, Wang, Chen et
al. 2005, Tan, Raychaudhuri et al. 2007)
Detailed cell cycle analysis revealed that the expression of FOXM1 protein
increases during G1 and S phases, reaching a maximal level in G2/M transition
(Korver, Roose et al. 1997, Laoukili, Kooistra et al. 2005). Despite FOXM1 mRNA
and protein being expressed throughout the cell cycle, its transcriptional activity is
tightly regulated in a cell cycle-dependent manner. Several important proliferation
and anti-proliferation signals regulate FOXM1 transcriptional activity involving post-
translational modifications and protein-protein interactions. The G1 phase regulator
CYCLIN D1/CDK4 complex strongly and indirectly activates FOXM1 by releasing the
TAD from RB repression and by the release of the TAD by the NRD repression. The
complexes CYCLIN E/CDK2, CYCLIN A/CDK2 and CYCLIN A/CDK1 also
phosphorylate and activate FOXM1 (Fig. 1.9), and the M phase associated CYCLIN
B1/CDK1 complex may also phosphorylate and activate FOXM1. The Ras-Raf-MEK-
ERK signalling pathway has been reported to regulate FOXM1 at multiple levels
including nuclear localization, protein expression, and transcriptional activity.
Antagonistically to these proliferation signals, the tumour suppressor ARF
(Alternative Reading Frame) interacts with FOXM1 to prevent its transactivation and
the tumour suppressor RB directly binds to the NRD and represses indirectly its TAD.
The cyclin dependent kinases inhibitors p16INK4a, p21Cip1 and p27Kip1 also repress
FOXM1 through the inhibition of CYCLIN D1/CDK4, CYCLIN E/CDK2 and CYCLIN
A/CDK2. Moreover, glycogen synthase kinase-3α targets the TAD of FOXM1 and
abolishes its transactivation (Wierstra and Alves 2006).
49
Figure 1.10 Cell cycle-dependent phosphorylation of FOXM1. Cell cycle-dependent regulation of FoxM1. FoxM1 protein expression (legend on the left) increases in late-G1. Upon mitogenic stimulation, Cyclin D/Cdk4, 6 and Cyclin E/Cdk2 inactivate pRb and relieve FOXM1 inhibition from pRb allowing the cells to progress into S phase. Increased FoxM1 transcriptional activity (legend on the right) in G2/M correlates with its hyperphosphorylation (Laoukili et al 2007).
protein protein
phosphorylation
50
1.6.3 FOXM1 function
1.6.3.1 Cell cycle
So far, the best described function of FOXM1 is its role in cell growth. It
stimulates proliferation by promoting G1/S and G2/M transitions (Leung, Lin et al.
2001, Wang, Hung et al. 2001, Laoukili, Kooistra et al. 2005, Wierstra and Alves
2007). The use of MEF foxm1-/- and siRNA interference demonstrated that FOXM1
is necessary for the expression of CDC25A, phosphatase required to activate CDK2
kinase activity during G1 and S phases. FOXM1 also facilitates CDK2 activation by
induction of SKP2 and CKS1 expression, which induces cdk inhibitors degradation by
the proteasome. In addition, CDC25A and cdk inhibitors regulate RB phosphorylation
and E2F release, which in turn stimulate transcription and cell cycle progression. For
the progression from G2 to M phase, FOXM1 controls the expression of CDC25B
and CYCLIN B1 and upregulates PLK, SURVIVIN and AURORA B during the mitotic
phase (Wang, Chen et al. 2005). Due to its role in mitosis, depletion of FOXM1 has
dramatic consequences such as aneuploidy and polyploidy, failure of the prophase
stage, misalignment of chromosomes at metaphase or mitotic spindle defect
(Laoukili, Kooistra et al. 2005, Wang, Chen et al. 2005, Wonsey and Follettie 2005).
1.6.3.2 Regenerative cell proliferation
In addition to its role in cell growth, FOXM1 is important for tissue repair. It was
shown that transgenic mice, with tissue-specific FOXM1 expression, display
accelerated cell proliferation following partial hepatectomy, liver or lung injury (Ye,
Holterman et al. 1999, Wang, Hung et al. 2001, Costa, Kalinichenko et al. 2003,
Kalinichenko, Gusarova et al. 2003). In contrast, regenerative cell proliferation is
reduced in mice with hepatocyte-specific and endothelial cell-specific FOXM1 knock-
out (Wang, Kiyokawa et al. 2002, Zhao, Gao et al. 2006).
51
1.6.3.3 Senescence
In agreement with FOXM1 importance in cell proliferation, MEF foxm1-/- and
cells from mice with pancreas-specific knock-out display senescence (Wang, Chen et
al. 2005, Zhang, Ackermann et al. 2006, Li, Smith et al. 2008, Park, Carr et al. 2009,
Zeng, Wang et al. 2009). The role of FOXM1 in carcinogenesis has also been
investigated and demonstrated that FOXM1 depletion sensitizes cells to oxidative
stress and senescence (Park, Carr et al. 2009). Furthermore, it was recently reported
that FOXM1 plays a role in senescence inhibition through transcriptional activation of
Bmi1 and inhibition of p27Kip1 (Li, Smith et al. 2008, Zeng, Wang et al. 2009).
1.6.3.4 Apoptosis
FOXM1 has recently been demonstrated to regulate apoptosis. It was first
reported that MEF FOXM1 knock-out and pancreas-specific FOXM1 depleted show
an increase in apoptosis (Zhang, Ackermann et al. 2006, Tan, Raychaudhuri et al.
2007, Bhat, Halasi et al. 2009). In addition, FOXM1 inhibition by thiazole antibiotic,
thiostrepton, siomycin A or ARF peptide inhibitor induce apoptosis in human cancer
cells and SV-40 transformed human lung fibroblasts (Kalinichenko, Major et al. 2004,
Radhakrishnan, Bhat et al. 2006, Kwok, Myatt et al. 2008, Bhat, Halasi et al. 2009).
Although studies have demonstrated a correlation between FOXM1 inhibition and
apoptosis, molecular mechanisms are not yet completely clarified.
1.6.3.5 DNA damage
The observation of aneuploidy and polyploidy in FOXM1 deficient cells indicates
a requirement of FOXM1 in chromosome stability and integrity. Consistent with this,
the percentage of DNA breaks increased when FOXM1 was depleted by knock-out
and siRNA interference (Tan, Raychaudhuri et al. 2007). FOXM1 is likely to have a
role in DNA damage but it is still elusive. A study has shown FOXM1 accumulation
promoted by CHK2 following IR, UV and etoposide, and a role in regulation of DNA
repair genes, including XRCC1 and BRCA2 (Tan, Raychaudhuri et al. 2007). In
52
addition, exogenous FOXM1 promotes cisplatin resistance in breast cancer cells
(Kwok, Peck et al. 2010).
1.6.3.6 Angiogenesis
Histological studies showed strong FOXM1 staining in gastric tumours and
lymph node metastases. The manipulation of FOXM1, by overexpression or
silencing, demonstrated that FOXM1 promotes cell growth and angiogenesis via the
modulation of genes involved in the degradation of the extracellular matrix and
angiogenesis such as uPA (urokinase-type kinase plasminogen activator), uPAR
(urokinase-type kinase plasminogen activator receptor), MMP-2 (matrix
metalloproteinase 2), MMP-9 (matrix metalloproteinase 9) and VEGF (vascular
endothelial growth factor) (Wang, Banerjee et al. 2007, Li, Zhang et al. 2009). This
finding was confirmed by the discovery of FOXM1 binding sites in VEGF and MMP2
promoters (Dai, Kang et al. 2007, Zhang, Zhang et al. 2008).
In summary, this tight antagonistic regulation of FOXM1 may require control to
exclude aberrant regulation of FOXM1 downstream target genes and their functions
that could result in tumourigenesis (Fig. 1.10).
53
Figure 1.11 FOXM1 functions. FOXM1 regulates a wide range of genes involved in key biological processes. FOXM1 regulates genes involved in G1/S and G2/M transitions as well as in genomic integrity leading to cell cycle progression and DNA repair. FOXM1 also regulates genes involved in apoptosis, metastasis and blood vessels formation. Deregulation of FOXM1 and its downstream targets enhance cell cycle progression, DNA repair, survival and angiogenesis and participate to the initiation and development of cancers.
1.7 FOXM1 IN CANCER
FOXM1 has been shown to be overexpressed in an extensive number of human
cancers, including gastric, cervical, breast, epidermal keratinocyte, lung, prostate,
colon and hepatocellular carcinomas (Kalinichenko, Major et al. 2004, Pilarsky,
Wenzig et al. 2004, Chandran, Ma et al. 2007, Yoshida, Wang et al. 2007,
Gialmanidis, Bravou et al. 2009, Zeng, Wang et al. 2009, Teh, Gemenetzidis et al.
2010, Kretschmer, Sterner-Kock et al. 2011). Particularly, it has been reported that
FOXM1 expression level increased with tumour grade and was inversely correlated
with patient survival (Kalin, Wang et al. 2006, Liu, Dai et al. 2006). Furthermore, the
chromosome band 12p13 where FOXM1 is located is frequently amplified in cervical,
head and neck carcinomas (Willem and Mendelow 1997, Sato, Kobayashi et al.
54
2001). Mice models highlighted the functional role of FOXM1 in tumour initiation and
progression. The knock-out of FOXM1 before liver cancer induction decreased liver
tumour development, and the knock-out of FOXM1 before lung and colon cancers
induction reduced the size and the number of adenocarcinomas (Kalinichenko, Major
et al. 2004, Kim, Ackerson et al. 2006, Yoshida, Wang et al. 2007). Similarly,
anchorage-independent growth on soft agar and tumour formation in nude mice were
increased by FOXM1 overexpression indicating that FOXM1 enhances tumour
development (Wang, Park et al. 2011). The overexpression of FOXM1 in mice model
increased the invasion capacity of glioma cells indicating that FOXM1 has a critical
role in metastasis (Dai, Kang et al. 2007, Raychaudhuri and Park 2011).
1.7.1 FOXM1 in breast cancer
A pioneer study in 2005 has revealed that FOXM1 mRNA expression level is
significantly overexpressed in 194 infiltrating ductal carcinomas, but not in
untransformed breast epithelial tissues. Furthermore, RT-qPCR data showed a
positive correlation between FOXM1 transcript levels and the stage of breast cancer
disease (Wonsey and Follettie 2005).
FOXM1 was identified among genes associated with high histological grade in
ERα positive breast cancers. During a survey of a panel of 16 different breast cell
lines, FOXM1 correlates with ERα at mRNA and protein levels (Madureira, Varshochi
et al. 2006). FOXM1 silencing results in a significant decrease of ERα expression
levels in ERα positive breast cancer cells. Conversely, ectopic expression of FOXM1
led to an upregulation of ERα transcript and protein levels. Furthermore, chromatin
immunoprecipitation assay identified FOXM1 binding site on ERα promoter region
(Madureira, Varshochi et al. 2006). Taken together, these data indicate FOXM1 as a
physiological regulator of ERα in breast carcinomas. Moreover, FOXM1 levels predict
early metastatic relapse for endocrine dependent breast cancers (Yau, Wang et al.
2011).
In addition to ERα, FOXM1 is associated with HER2 receptor. FOXM1 and
HER2 mRNA and protein levels expression correlate in breast cancer cell lines and
patient samples. Consistently, investigations of mammary epithelium targeted HER2
mouse tumours resulted in an increase of FOXM1 expression (Francis, Myatt et al.
55
2009). Data from breast cancer cell lines demonstrated that HER2 directly regulates
FOXM1 in HER2 overexpressing breast cancers.
While there is no biomarker identified for patients who are oestrogen and
progesterone receptor negative and exhibit low HER2 expression breast cancer,
these patients are treated with chemotherapy and have poorer prognostic than
receptor positive cancer patients. However, a recent study has shown that FOXM1 is
found elevated by DNA copy number alterations in triple negative breast cancer,
suggesting that targeting FOXM1 would be beneficial regardless the receptor status
(Han, Jung et al. 2008).
Taken together, FOXM1 inhibition could be a new strategy to treat breast
cancer patients of any types.
1.7.2 Development of FOXM1 inhibitors
FOXM1 is an attractive target for targeted therapy because it has a key role in
many biological processes and is overexpressed in a majority of cancers (Wang,
Ahmad et al. 2010). This notion is supported by studies using RNA interference to
knock-down FOXM1 expression. Depletion of FOXM1 in breast cancer cells lead to
inhibition of cell growth, clonogenicity, migration and invasion (Wonsey and Follettie
2005). In addition, FOXM1 silencing reduced cell proliferation and anchorage
dependent cell growth on soft agar in several prostate and lung cancer cell lines
(Kalinichenko, Gusarova et al. 2003, Kalin, Wang et al. 2006).
Consistent with this, a study in 2004 revealed that FOXM1 is essential for
initiation of carcinogen-induced liver tumours since liver cells with FOXM1 conditional
depletion fail to proliferate and are resistant to liver cancer development
(Kalinichenko, Major et al. 2004). Further results reported that ARF26-44 peptide can
bind and inhibit FOXM1 transcriptional activity resulting in inhibition of cell
proliferation and induction of apoptosis (Gusarova, Wang et al. 2007).
Within the last ten years, an emerging class of naturally occurring thiostrepton
group of antibiotics has shown a range of antibacterial, anti-parasitic and anti-cancer
properties. In 2008, evidence showed that thiostrepton antibiotic selectively reduced
FOXM1 expression resulting in breast cancer cell death. Furthermore, a study using
a cell-based screening assay system identified another member of the family of
56
antibiotics, siomycin A, as a potent inhibitor FOXM1 transcriptional activity. The
efficacy of siomycin A and thiostrepton was studied in neuroblastoma, liver, leukemia,
melanoma and breast cancer and results showed that the antibiotics induced
apoptosis (Bhat, Zipfel et al. 2008, Kwok, Myatt et al. 2008, Bhat, Halasi et al. 2009).
Siomycin A has been shown to specifically reduce FOXM1 transcriptional activity,
while the mechanism of action of thiostrepton still needs to be clarified.
Taken together, these data suggest that targeting FOXM1 is a promising
strategy for treating breast cancer and many other cancers. Moreover, studies have
shown FOXM1 involvement in resistance to targeted therapy and chemotherapy. It is
possible that inhibiting FOXM1 in combination with anti-cancer therapies will improve
the efficacy of currently available treatments.
1.8 HYPOTHESES AND OBJECTIVES: FOXM1 as a therapeutic strategy to overcome drug resistance
Several members of the forkhead family have been found to be involved in
diverse mechanisms of drug resistance. Notably, FOXO and FOXM1 members were
associated with targeted therapies and chemotherapies resistance.
FOXO proteins play an important role in protection of cells against genotoxic
and environmental stresses. Although FOXO3A activation by anti-cancer drugs
induces cell cycle arrest and apoptosis, chronic activation by doxorubicin induces the
transcriptional expression of MDR1 leading to increased drug efflux ability, which can
render cancer cells resistant to drug therapy (Hui, Francis et al. 2008). Activated
FOXO3A can also contribute to the development of resistance to HER2 targeted
therapies by increasing AKT/PI3K activity through the induction of PIK3CA
expression and promotion of cell survival (Chen, Gomes et al. 2010). Another FOXO
member, FOXO1 protein serves as a protector against oxidative stress and its
contribution to drug resistance was highlighted during human pregnancy, when the
human endometrial stromal cells are exposed to high fluctuations in oxygen levels.
Through the induction of the expression of manganese superoxide dismutase
(MnSOD), FOXO1 confers resistance to oxidative stress-induced apoptosis (Kajihara,
Jones et al. 2006). Furthermore, FOXO1 is highly expressed in paclitaxel-resistant
ovarian cells and enhanced by paclitaxel exposure. FOXO1 overexpression was
57
frequently observed in tissue samples from paclitaxel-resistant patients compared to
paclitaxel-sensitive patients. FOXO1 silencing rendered chemoresistant cells
sensitive to paclitaxel-induced apoptosis (Goto, Takano et al. 2008). In addition to
taxane resistance, FOXO1 has been involved in breast cancer resistance to
anthracyclines. A reporter assay showed that FOXO1 stimulates the transcription of
MDR1 gene expression in MCF-7 cells. MDR1 expression and doxorubicin resistance
in MCF-7 resistant cells was reversed by FOXO1 silencing indicating that FOXO1
expression is crucial for chemoresistance (Han, Cho et al. 2008).
In vitro studies showed that ectopic expression of FOXM1 confers breast cancer
cells resistance to trastuzumab and paclitaxel. Resistance to the growth inhibitory
effect of trastuzumab has been accounted by its ability to maintain low level of
p27Kip1, preventing its accumulation and cell cycle arrest. Moreover, FOXM1
transcriptionally activates the expression of STATHMIN, which inhibits the
polymerization of the microtubules in response to the taxane agent paclitaxel (Carr,
Park et al. 2010). An additional study demonstrated an increased FOXM1 protein
expression in cisplatin resistant breast cancer cells (Kwok, Peck et al. 2010). FOXM1
contribution to cisplatin resistance is thought to be due to its role in DNA repair.
However, the role and detailed mechanisms of FOXM1 involvement in DNA repair
and resistance have not yet been elucidated. Moreover, it was recently reported that
overexpression of FOXM1 partially protects osteocarcinoma cells from apoptosis
induced by thiazole antibiotic (Bhat, Halasi et al. 2009). The emerging evidence from
in vitro and in vivo studies demonstrate that FOXM1 plays an important role in
initiation and progression of cancer by the regulation of many target genes and cross-
talking with multiple signalling pathways (Kalin, Ustiyan et al. 2011). Therefore,
FOXM1 signalling pathway is a promising therapeutic target and the development of
agents targeting FOXM1 is likely to have a great impact for the treatment of drug
resistant breast cancer.
58
1.8.1 FOXM1 regulation and role in tamoxifen sensitivity and resistance
Due to an increase of evidence in clinical resistance to a wide range of targeted
therapeutic and chemotherapeutic agents, the development of novel drugs to
overcome drug resistance is needed. Tamoxifen is the main endocrine treatment
used for ERα positive breast cancer patients. However, 70 % of patients that initially
respond to tamoxifen become resistant after long term treatment. ERα is a strong
proliferative factor activating the expression of a wide range of genes encoding
cytokines and factors associated with immune response, signal transduction, cell
migration and cytoskeleton regulation. Data has showed that deregulation of ERα at
transcriptional or posttranslational level can elicit anti-oestrogen resistance. Previous
work in the laboratory reported that FOXM1 and ERα correlate at mRNA and protein
levels in a panel of breast cancer cell lines. Further results showed that FOXM1
activates ERα transcriptional expression directly through the binding of forkhead site
within ERα promoter in breast cancer cell lines (Madureira, Varshochi et al. 2006).
Previous data also showed that FOXM1 is often regulated through a positive
feedback loop with genes involved in proliferation such as CYCLIN B1 and PLK
(Leung, Lin et al. 2001, Laoukili, Kooistra et al. 2005, Fu, Malureanu et al. 2008).
These co-regulations occur amongst genes involved in cell cycle, which could lead to
uncontrolled proliferation and drug resistance of cancer cells. Therefore, it is
important to investigate FOXM1 regulators. This thesis examines the regulation of
FOXM1 by ERα, given that FOXM1 is frequently overexpressed in breast cancer, and
ERα deregulation leads to the development of anti-oestrogen resistance.
Furthermore, this thesis examines reversing anti-oestrogen resistance by
downregulating FOXM1 as therapeutic approach.
1.8.2 FOXM1 regulation and role in chemotherapy sensitivity and resistance
Hormone receptor negative patients can only be administrated
chemotherapeutic agents. Identification of potential biomarkers is urgently needed to
elucidate novel therapies and improve the overall survival rate. FOXM1 has been
59
identified in a high resolution array to be amplified in hormone receptor negative
breast cancer tissue patients suggesting that FOXM1 could be a potential biomarker
in hormone receptor negative breast cancer (Han, Jung et al. 2008). FOXM1 target
genes are involved in cell proliferation; oxidative stress and DNA repair processes.
Recent data identified FOXM1 as a mediator of cisplatin resistance. FOXM1
involvement in cisplatin resistance is thought to be due to an increase in DNA repair
but the detailed molecular events have not been clarified yet (Kwok, Peck et al.
2010). Further studies revealed that FOXM1 is stabilized by DNA damage agents
through the phosphorylation by checkpoint kinase, CHK2, and leading to the
regulation of genes involved in homologous recombination DNA repair mechanism
(Tan, Raychaudhuri et al. 2007). Cisplatin is a strong alkylating agent given to treat a
variety of cancers, but is limited by its toxicity profile. Nowadays, anthracyclines,
doxorubicin or epirubicin, are the most widely prescribed chemotherapeutic agents.
For nearly thirty years, the anthracyclines, doxorubicin and epirubicin, have been
pivotal in the management of early stage breast cancer, particularly in hormone
receptor negative cases (Boér 2010). As emerging evidence has shown that FOXM1
is involved in drug resistance and may in fact be a potential biomarker, this thesis
investigates the involvement and regulation of FOXM1 in epirubicin-sensitive and –
resistant breast cancer.
60
CHAPTER 2 MATERIAL AND METHODS
61
2.1 CELL CULTURE
2.1.1 Cell lines
The human breast carcinoma MCF-7, ZR-75-1, and MDA-MB-231, MDA-MB-453
cell lines originated from the American Type Culture Collection, were obtained from
the Cancer Research UK Cell Line lab (CRUK, Clare Hall, UK), in which they were
tested and authenticated, and maintained in Dulbecco’s Modified Eagle Medium
(DMEM) supplemented with 10 % Foetal Calf Serum (FCS), 2 mM glutamine, and
100 units/ml penicillin/streptomycin at 37 °C in an atmosphere of 10 % CO2.
The COS-1 cells were derived from kidney cells and grown in DMEM
supplemented with 10 % Foetal Calf Serum (FCS), 2 mM glutamine, and 100 units/ml
penicillin/streptomycin at 37 °C in an atmosphere of 10 % CO2.
2.1.2 Stably transfected cell lines
Previously in our laboratory, parental MCF-7 cells were stably transfected with
the N-terminal deleted FOXM1 fragment (Park, Wang et al. 2008) and the full-length
FOXM1 in pcDNA3 expression vector and the transfection was maintained by DMEM
supplemented with 1 µg/ml puromycin selection marker (Invitrogen, Paisley, UK).
2.1.3 Knock-out cells
The mouse embryonic fibroblasts (MEF) were derived from wild-type, p53-/- and
p21Cip1-/-. The MEF wild-type (wt) and foxm1-/- were kindly provided by Pr René H.
Medema (Department of Medical Oncology, University Medical Center Utrecht, The
Netherlands). All cells were grown in humidified atmosphere 10 % CO2 at 37 °C and
in DMEM supplemented with 10 % FCS, 2 mM glutamine, and 100 units/ml
penicillin/streptomycin.
62
The wild-type 48BR (human fibroblast) primary skin fibroblasts were a generous
gift by Dr Penny Jeggo (University of Sussex, UK). The human fibroblasts deficient in
NBS1 (NBS1‐LBI) were kindly given by Veronique Smiths (Unidad de investigacion,
Hospital Universitario de Canarias, Spain). All cells were grown in humidified
atmosphere 10 % CO2 at 37 °C and in DMEM supplemented with 10 % FCS, 2 mM
glutamine, and 100 units/ml penicillin/streptomycin.
2.1.4 Drug resistant cell lines
The tamoxifen 4-OHT resistant MCF-7TAMR4 cells have been kindly given by
Anne Lykkesfeldt (Institute of Cancer Biology, Denmark), and their growth conditions
previously characterized and described [Lykkesfeldt, 1994 #229; Madsen, 1997
#230]. Briefly, a clone of MCF-7TAMR cells were established by two series of one
week treatment with 10-6 mol/L 4-OHT (OHT) and the resistant cells were
continuously propagated with 10-6 mol/L OHT. The MCF-7 and derivatives cells were
exposed to ER ligands: 10-8 mol/L estradiol (E2), 10-6 mol/L 4-OHT (OHT) or 10-7
mol/L ICI182780 (ICI) (prepared in ethanol), or only ethanol (vehicle control) for the
indicated times prior to harvesting. For steroid starvation, these cells were cultured in
phenol-free DMEM/F-12 containing 5 % double charcoal-stripped FCS.
The MCF-7EPIR cell line is an epirubicin resistant cell line derived from parental
MCF-7 cells. Previously in our laboratory, MCF-7 cells were subjected to a gradual
concentration of epirubicin until cells acquire resistance up to 10 µmol/L of epirubicin
(Pfizer, UK). MCF-7EPIR cells were maintained in 1 µmol/L of epirubicin. Prior
experiments, epirubicin was removed for 24 h and then cells were treated with
epirubicin 1 µmol/L for the indicated times before harvesting. For RNA interference,
the cells were treated with epirubicin 24 h after transfection. For ATM inhibition, cells
were treated with Ku-55933 at 10 µmol/L for 24 h alone or in combination with 1
µmol/L epirubicin.
63
2.1.5 Cell line maintenance
Cells were grown and split at approximately 90 % twice a week. Media was
aspirated and the cell monolayer was rinsed once with 1X PBS and then detached
using 1X trypsin-EDTA mix. After centrifugation at 1200 rpm for 5 min, cells were
resuspended in the appropriate media and seeded into an appropriate flask or dish.
For long term maintenance, cells were detached from the flask or dish by the
addition of 1X trypsin-EDTA, spun 1200 rpm for 5 min. The supernatant was
discarded and the cell pellet was resuspended in FCS with 10 % dimethyl sulphoxide
(DMSO) at 1 million cells/ml and 1 ml was transferred per cryotube and slowly frozen
at -80 °C for 2 days before being transferred to storage in liquid nitrogen. For
defrosting cells, cryotubes were placed for 1min in a waterbath at 37 °C and the
defrosted solution was added to complete media and spun at 1200 rpm for 5 min. the
DMSO containing supernatant was removed and cell pellets were resuspended in
fresh supplemented medium in flask or dish.
2.1.6 Chemicals
Tamoxifen was maintained as a stock solution at 2 mM at -20 °C and diluted in
fresh media prior to treatment (Pfizer, UK). ICI182780 and estradiol were dissolved
in ethanol and stored -20 °C at a concentration of 10-2 M (SigmaAldrich, UK).
Epirubicin (2 mg/ml in 0.9 % sodium chloride) was obtained from Pfizer UK and
stored at 4 °C. Ku-55933 was dissolved in ethanol and stored at -20 °C at a
concentration of 10 mmol/L (Tocris Bioscience, UK).
64
2.2 PROTEIN ANALYSIS
2.2.1 Preparation of total protein lysates and determination of protein concentration
Whole cell extracts was prepared by harvesting cells using 1X trypsin-EDTA and
spinning at 1200 rpm for 5 min to obtained cell pellets. The supernatant was
discarded and cell pellets were washed in 1X PBS (Phosphate Buffered Saline) and
spun for an additional 5 min at 1200 rpm. The supernatant was discarded and cell
pellets were frozen at -80 °C until lysis was performed. Frozen pellets were lysed in a
lysis buffer containing 0.1 % Triton X100, 150 mM NaCl2, 50 mM Tris-HCl (pH 7.8), 1
mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride and protease
inhibitors (“Complete protease inhibitor mixture, as instructed by the manufacturer,
Roche Applied Sciences, UK) on ice for 30 min (Table 2.1). Supernatant were
collected after microcentrifugation at 13000 rpm at 4 °C for 10 min and protein
concentration was measured using Bio-Rad Dc protein assay (Bio-Rad Laboratories,
CA, USA) as instructed by the manufacturer. 20 μl of reagent S was added to 1 ml of
reagent A. A standard curve was established by assaying 5 dilutions of a protein
standard within the range of 0.2 mg/ml to 1.5 mg/ml protein. 25 μl of reagent A was
added to 2 μl of each dilution and mixed with 200 μl of reagent B. After 15 min,
absorbance was read at 700 nm. The protein concentration of the samples was
determined by multiplying the absorbance of the sample by the standard curve’s
regression coefficient.
2.2.2 Western blotting or sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
Protein expressions were determined using SDS-PAGE gels made using a 37.5
% (w/v) acrylamide/bis stock solution, Tris-HCl and, 25 % of ammonium persulphate
and tetramethylethylenediamine (TEMED). SDS-PAGE gels consist of a lower
resolving gel and an upper stacking gel. The percentage of resolving gel used
depends on the size of the protein of interest. The protein extracts were diluted at 20
μg in 2X SDS loading buffer (4 % (w/v) SDS, 62.5 mM Tris-HCL (pH 6.8), 1 % (v/v)
65
glycerol, 0.01 % (w/v) bromophenol blue, 10 % (v/v) β-mercaptoethanol) and boiled
at 100 °C for 5 min. Samples were then run at a constant voltage of 100V in running
buffer (Table 2.1). Proteins were separated alongside a Novex® Sharp Pre-Stained
protein standard (Invitrogen, Paisley, UK) to identify the molecular weight of the
separated proteins. Subsequently, proteins were transferred to nitrocellulose
membranes (Whatman® Protran®) using a transfer buffer at 90 V for 1 h (Table 2.1).
After the transfer, membranes were saturated with a 5 % blocking solution (Bovine
Serum Albumin or Milk) diluted in TBST for 1 h at room temperature (RT) (Table 2.1).
The primary antibody was diluted at 1:1000 in the blocking solution previously used
and incubated overnight at 4 °C, the membranes were then washed three times with
TBST (Table 2.1) for 15 min and incubated either with peroxidase-conjugated
secondary antibody against mouse or rabbit IgG at 1:5000 dilution in TBST for 45 min
at RT. After the TBST washing steps, membranes were incubated with Western
Lightning® ECL (chemiluminescence peroxidase substrate) according to the
manufacturer’s instructions (Perkin Elmer, UK) and a signal was detected using
Hyperfilm ECL (GE healthcare) on SRX-101A x-ray developer.
Reagent Recipe
Western Blotting
1X TG 25 mM Tris, 192 mM Glycine, pH 8.3
TBS 10X 24.23 g Tris HCl, 80.06 g NaCl add up to 1 L with ddH20 and adjust pH to 7.6 with concentrated HCl
TBST 100 ml of TBS 10x, 890 ml ddH20, 10 ml Tween 10 % (v/v)
Lysis buffer 50 mM Tris pH 7.5, 150 mM NaCl, 0.10 % Triton x100 , 10 mM NaF, 1 tablet protease inhibitors (Roche)
Running buffer 1x TG, 0.1 % SDS
Transfer buffer 100 ml TG 10x, 800 ml ddH20, 100 ml Ethanol
Blocking solution 5 % (w/v) BSA, TBST, 0.02 % (w/v) sodium azide
Chromatin Immunoprecipitation
TSE I buffer 0.1 % (w/v) SDS, 1 % (v/v) Triton X-100, 2mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl
Buffer I 0.25 % (v/v) Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES pH 6.5
Buffer II 200 mM NaCl, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES pH 6.5
Lysis buffer 1 % SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, 1 tablet protease inhibitors
Table 2.1 SDS-PAGE and ChIP buffers
66
Antibody Company Reference Specie kDa
Transcription factors and coactivators
FOXM1 (WB, ChIP) Santa Cruz sc-502 Rabbit 100 FOXM1 (IF) Santa Cruz sc-500 Rabbit 100 ERα Santa Cruz sc-543 Rabbit 66
AIB-1 BD transduction laboratories
611105 Mouse 160
P-p53 (Ser15) Cell Signaling 9284 Rabbit 53 p53 Santa Cruz Pab1801 Mouse 53
Histones and histones modificators
HDACI Santa Cruz sc-6299 Goat 55 HDACII Abcam ab7029 Rabbit 60 Acetyl-H3 (ChIP) Upstate 06-599 Rabbit 10 Acetyl-H4 (ChIP) Upstate 06-866 Rabbit 17
Cell cycle regulators
Cyclin A Santa Cruz sc-239 Mouse 54 Cyclin B1 Santa Cruz sc-752 Rabbit 60 Cyclin D1 Santa Cruz sc-246 Mouse 37 CDK2 Abcam ab2363 Mouse 34 CDK4 Cell Signaling DCS156 Mouse 30 p21 Santa Cruz sc-6246 Mouse 21 Cdc25b Abcam ab2358 Mouse 90 PLK Santa Cruz sc-17783 Mouse 66 pS2 Santa Cruz sc-28925 Rabbit 7-12 E2F1 (WB, ChIP) Santa Cruz sc-193 Rabbit 60 P-pRB (Ser807/811) Cell Signaling 9308 Rabbit 110 pRB (ChIP) BD Pharmigen 554136 Mouse 110
DNA damage and checkpoint markers P-ATM (Ser1981) Upstate MAB3806 Mouse 370 ATM Calbiochem Ab-3 Rabbit 370 P-Chk1 (Ser345) Cell Signaling 133D3 Rabbit 59 Chk1 Santa Cruz sc-8408 Mouse 56 P-Chk2 (Thr68) Cell Signaling C13C1 Rabbit 62 Chk2 Upstate clone 7 Mouse 67 P-H2AX (Ser139) Upstate clone JBW301 Mouse 17 H2AX Cell Signaling 2595 Rabbit 17 NBS1 Cell Signalling 3002 Rabbit 95
Cell death markers
PARP Cell Signaling 9542 Rabbit 89,116
Ubiquitous proteins
Beta-Tubulin Santa Cruz (H-235)sc-9104 Rabbit 57
Table 2.2 Antibodies for western blotting and ChIP
67
2.3 PULL-DOWN using biotin-labelled oligonucleotides
Nuclear and cytoplasmic extracts were prepared using NE-PER® nuclear and
cytoplasmic extraction kit (Thermo Scientific, Rockford, USA). Briefly, cells were
collected using trypsin and centrifuged to obtain dry pellet. CER I buffer (cytoplasmic
extraction reagent) was added to the pellet, incubated for 5 min and centrifuge at
maximal speed. The supernatant (cytoplasmic fraction) was transferred to a new
eppendorf and stored at -80 ºC until use. The insoluble pellet was then resuspended
in NER buffer (nuclear extraction reagent) and incubated on ice for 40 min. After the
same process of centrifugation, the nuclear fraction was transferred to a new
eppendorf and store at -80 ºC until use.
The biotinylated oligonucleotides, oestrogen response element (ERE) (ERE-wt
5’-GCCGATTGGCGACGTTCCGTCACGTGACCTTAACGCTCCGCCGGCG-3’, 5’-
CGCCGGCGGAGCGTTAAGGTCACGTGACGGAACGTCGCCAATCGGC-3’), or
(mERE3 5’-GCCGATTGGCGACGTTCCGTAACGTTACGTTAACGCTCCGCCGGC-
3’, 5’-CGCCGGCGGAGCGTTAACGTAACGTTACGGAACGTCGCCAATCGGC-3’),
were firstly annealed and bound to streptavidin beads on a rotator for 2 h at RT. 50
µg of protein extract were added with or without an excess of unlabelled competitor
oligonucleotides and incubated for 1 h at 4 °C on the rotator. Beads were then
washed with three times 1X PBS, loading buffer was added. Beads were boiled at
100 °C for 5 min and analysed by western blotting the supernatant.
2.4 IMMUNOPRECIPITATION AND IMMUNOBLOTTING
Cells were harvested and lysed as described in the protein analysis section.
While the cells pellets were being extracted, 20 µl of dynabeads per reaction
(Invitrogen, Paisley, UK) were washed with 1X PBS and incubated for 1 h 4 ºC with
the appropriate antibody. After centrifugation at 14 000 rpm, protein lysates were
incubated with the complexes beads/antibodies for 2 h at 4 ºC. For immunoblot
analysis, the immunoprecipitated samples were diluted in 2X SDS-loading buffer,
boiled and run on SDS-PAGE gels. After the transfer, membranes were blocked,
incubated with primary antibody overnight 4ºC and then with the relevant peroxidase-
68
conjugated secondary antibody for 1 h RT and visualised using Western Lightning®
ECL
2.5 CHROMATIN IMMUNOPRECIPITATION (ChIP)
2.5.1 Beads preparation
The day before chromatin immunoprecipitation was performed, 20 µl of
dynabeads per condition (Invitrogen, Paisley, UK) were washed with TSE I buffer
(Table 2.1) three times using a magnetic rack to discard the supernatant and were
resuspended in 20 µl of TSE I buffer. One microgram of antibody and control
immunoglobulin were incubated with the beads overnight on the rotator at 4 °C
(Table 2.2).
2.5.2 Cells preparation
The cells were seeded in 10 cm dish to obtain 90 % confluency prior
experiments. About 10 million of cells were cross-linked by adding 270 µl of 37 %
formaldehyde to the 10 ml of cell medium (1 % formaldehyde) and were incubated at
37 °C for 10 min. Under the chemical hood, formaldehyde/medium mix was
discarded and cells were gently washed twice with cold 1X PBS. One millilitre of the
mix containing 100 mM Tris-HCl pH 9.4 and 10 mM DTT was added to each 10 cm
dish to scrape the cells and transferred to an ice-cold sterile eppendorf. Cells were
then centrifuged at 5 000 rpm for 5 min. Pellets were sequentially washed with 1 ml
of 1X PBS, ChIP buffer I and ChIP buffer II (Table 2.1) to obtain chromatin.
69
2.5.3 Sonication
After these washing steps, cell pellets were resuspended in a ChIP lysis buffer
(Table 2.1), incubated on ice for 10 min and sonicated for 12 min at 30 sec intervals.
Optimisation of sonication times has been optimised with the different cell lines used.
The sonication time was determined by running DNA (chromatin) on 1 % DNA gel
electrophoresis and selecting time for which DNA size is between 100-500 bp.
2.5.4 DNA/beads-antibody incubation
After sequential washing steps, the lysates were microcentrifuged at 14000 rpm
for 10 min at 4 °C. The chromatin pellets obtained were resuspended in the TSE I
buffer, transferred to the beads-antibody complexes and incubated on the rotator for
2 h at 4 °C. Afterwards, the beads were washed with the TSE I buffer five times and
the DNA was eluted with a mixture of 1 % (w/v) SDS and 0.1 M NaHCO3 twice for 1 h
on the rotator.
2.5.5 DNA elution, purification and Polymerase Chain Reaction (PCR)
The samples were decrosslinked at 65 °C overnight and DNA was purified using
the QIAquick PCR Purification kit (Qiagen, Paisley, UK), as described in the
manufacturer’s instructions. After the purification, a Polymerase Chain Reaction
(PCR) was set up with primers designed using the ABI Primer Express software
(Table 2.3). 50 ng of total eluted DNA or 1% of eluted DNA (input) with 1 µM of a
forward and reverse primer specifically designed for each target gene (at the DNA
binding site tested and control site upstream), mixture of dNTP and mix reaction of
the DNA polymerase kit containing 10X buffer, Q solution, MnCl2 and Taq
polymerase as described by the manufacturer (Qiagen, Paisley, UK) were incubated
in a thermocycler (GeneAmp PCR system 9700, Applied Biosystems).
70
The controls performed include:
- primers for a region where the protein of interest is absent (negative control)
that were tested for each experiment (shown in the results).
- a non-template control that was included in each PCR reaction to spot
contamination (not shown in the results).
Figure 2.1 DSB detection and repair model. I-PpoI cuts DNA (1) inducing chromatin structural change that initiates ATM activation. Activated ATM is recruited to DSBs and phosphorylate MRN proteins (2). Repair proteins such as XRCC4 are recruited to the DSB (3) (from (Berkovich, Monnat et al. 2007).
71
2.5.6 DNA gel electrophoresis
Agarose (SigmaAldrich, UK) was dissolved in the appropriate volume of 1X TAE
(Table 2.4) at the concentration of 1 % (w/v) and the mixture heated until fully
dissolved. Ethidium bromide was added once the mixture was allowed to cool.
Samples diluted in DNA loading buffer (Table 2.4) and DNA ladder was resolved and
DNA visualized under Ultra Violet light using UVIPro Platinum software.
Buffer Recipe
Tris Acetate EDTA (TAE 50X) 2 M Tris, 57.1 ml acetic acid, 0.2 M EDTA pH 8.0, ddH2O added until total volume 1 L
DNA Loading buffer
2.3 M sucrose and 100 mg Orange G made to 50
ml with ddH2O
Table 2.3 DNA gel electrophoresis buffers
2.6 RNA ANALYSIS
2.6.1 Total RNA extraction
Total RNA was isolated from cells using the RNeasy Mini kit (Qiagen, Crawley,
UK). The protocol was performed in line with the manufacturer’s instructions.
Frozen cell pellets were resuspended in 350 μl of RLT buffer (containing 10% β-
mercaptoethanol) and homogenised by pipetting. 350 μl of 70 % ethanol was added
and the total mixture was transferred to the provided column, which was placed in a 2
ml collection tube, and spun in a benchtop centrifuge at 10000 rpm for 30 secondes
(sec). The flow through was discarded and 700 μl of buffer RW1 was added to the
column and spun at 10000 rpm for 30 sec. Then, 500 μl of RPE buffer was added to
the column and spun at 10000 rpm for 30 sec. the low through was discarded and
the column was spun for an additional 10000 rpm for 30 sec to remove any waste of
the column. Next, the column was transferred to a clean sterile eppendorf tube. The
72
extracted RNA was eluted by the addition of 30 μl of RNase-free water to the centre
of the column and spinning for 1 min at 10000 rpm. The purity and concentration of
the RNA was determined using Nanodrop, which measures the spectrometric
absorption at 260 nm and 280 nm. RNA samples were then stored at -80 °C or
immediately used to make cDNA using first strand cDNA synthesis.
2.6.2 First strand cDNA synthesis
1 μg of total RNA was reverse transcribed into first strand cDNA using the
Superscript III first strand cDNA synthesis system (Invitrogen, Paisley, UK).
One microliter of random primers and 1 μl of 10 mM dNTPs mix (containing four
bases adenine, cytosine, guanine and thymine) were added to 1 μg of total RNA to
make a total volume of 14 μl using sterile RNase free water. The sample was heated
for 5 min at 65 °C and placed immediately on ice for 1 min. Four microliters of 5X first
strand buffer, 1 μl of 0.1 M DTT, 1 μl of RNaseOUT and 1 μl of the reverse
transcriptase Superscript III were then added to make a total volume of 20 μl. The
mixture was placed in a thermocycler (GeneAmp PCR system 9700, Applied
Biosystems) where they were incubated for 5 min at 25 °C for 5 min, then heated to
50 °C for 50 min. the reaction was terminated by heating the mixture at 70 °C for 15
min.
2.6.3 Primers
The following human and mouse gene-specific primer pairs were designed using
the ABI Primer Express software:
73
Table 2.4 Human and mouse gene-specific primer pairs for RT-qPCR and ChIP
Forward sequence Reverse sequence
Temp
(˚C)
ChIP primers (human):
FOXM1 (ERE) CCACTTCTTCCCCCACAAG CCGGAGCTTTCAGTTTGTTC 65
FOXM1 (E2F) CCACTTCTTCCCCCACAAG CCGGAGCTTTCAGTTTGTTC 65
FOXM1 (cont) CCACGCTTCCCCCACAAG CCGGAGCTTTCAGTTT 65
NBS1 (FHK) AATTAAAAATTTTCCTTATGTTGCTTT GGGCGCTTGCCCGCCACCTGGTGGTTGG 60
NBS1 (cont) GCTAGAGTGCAGTGGCATGA AAGATCAGCATGGGCAACAT 60
DAB1 TGCTGCTTTTTCTTCTTCTCC CTTCTTTCCCACCAAGTCTTC 64
Β-actin AACTCCATCATGAAGTGTGACG GATCCACATCTGCTGGAAGG 60
Gene expression primers (human):
FOXM1 TGCAGCTAGGGATGTGAATCTTC GGAGCCCAGTCCATCAGAACT 60
ERα CAGATGGTCAGTGCCTTGTTGG CCAAGAGCAAGTTAGGAGCAAACAG 60
E2F1 CTGAGACAACTTGAGGAAGAG TTTGAACCTGTACTAGCCAGTC 60
ATM AATATCCATTCACCGCAGCC CACAATTTGCCGTAGGTAGTATC 60
ATR AGTCCCAGCCAGTCTCTACTCA TGCCCATCCGGGACAA 60
NBS1 TTTTCAACCAGTTTTCCGTTACTTC ACACTGCGCGTATAAGCCAAT 60
MRE11 TGAGAACTGGCCTTCGATTCA GGAGCCCAGACAAGCATGAT 60
RAD50 TCCAAATCTTGTGGAAGTGCAT CTGCAAGCAGCCAGAACTTG 60
L19 TCTGGATGATGCTGTGCTACCT GGCCCACAGCTCAGACTGA 60
Forward sequence Reverse sequence
Temp
(˚C)
Gene expression primers (mouse):
Foxm1 TGCAGCTAGGGATGTGAATCTTC GGAGCCCAGTCCATCAGAACT 60
Nbs1 TGACAACCCGATAGAGGAGCAT TCTTGGCTCTCTGTCTGTCCAG 60
Mre11 TTCCCTCGGTGGGATTCAA ACACCCATCTGGCTGTCAGAA 60
Rad50 TAGCACACCAACACGTCGTA CAGTGCCTTCCTCCTCTTGT 60
Atm GCGCCACGCCTTGT CAAACGTTGCCTGAAT 60
L19 TACACCTTCCCACTTACTGA ATTCCTCCGACTCTTCCTTT 60
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2.6.4 Real-time quantitative PCR (RT-qPCR)
The specificity of each primer was determined using NCBI BLAST module. Real
time PCR was performed with ABI PRISM 7700 Sequence Detection System using
SYBR Green Mastermix (Applied Biosystems, Brackley, UK).
Transcript levels were quantified using the standard curve method, where 1 µg of
cDNA from each sample was mixed and diluted into serial dilutions (1/4, 1/16, 1/64,
1/256). L19, a non-regulated housekeeping gene, was used as an internal control to
normalise the input cDNA.
The reaction mix contained 2 µl of sample and 23 µl of of SYBR Green master
mix, primers and RNase-free water were added to a final volume of 25 µl. All
experiments were performed in triplicates.
2.7 DNA MANIPULATION
2.7.1 Plasmid amplification and extraction
The XL1-Blue competent cells were used to amplify plasmids. 50 µl of XL1-Blue
(per vector) were thawed on ice and 2 µl of plasmid was added and incubated on ice
for 30 min. The mixture competent cells and plasmid was heated at 42 ºC in a
waterbath for 45 sec and chilled on ice for 2 min prior incubation with 500 µl of SOC
medium for 1 h at 37 ºC. 100 µl was spread into warm LB-agar plates containing the
selective antibiotic and incubated at 37 ºC overnight.
Sixteen hours after incubation, individual colonies were grown overnight in 3 ml
of LB-broth supplemented with the corresponding antibiotic. The following day, 1 ml
of overnight culture was used for screening. Bacterial DNA was extracted using the
miniprep protocol (Qiagen, Crawley, UK) following the manufacturer’s instructions
and DNA was digested using restriction enzymes to verify the presence of the insert.
Positive clones were then grown overnight in 250 ml of LB-broth supplemented with
the antibiotic and plasmid was extracted with a maxiprep protocol from Qiagen.
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Reagent Recipe
Luria-Bertani (LB) medium 1% (w/v) tryptone, 1% (w/v) yeast extract, 1% (w/v) NaCl
LB-agar medium 1% (w/v) tryptone, 1% (w/v) yeast extract, 1% (w/v) NaCl
SOC media 2% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl, 5 mM MgSO4, 10 mM MgCl2, 0.4% (w/v) glucose
Ampicillin 1 g Ampicillin in 10 ml ddH2O (final concentration 100 mg/ml)
Ampicillin resistance selection media/agar Ampicillin added to LB medium/agar at final concentration 100 µg/ml
Table 2.5 Bacterial culture reagents
2.7.2 DNA mutation and sequencing
Mutagenesis of plasmids were performed using the Stratagene Quickchange
site-directed mutagenesis kit (Agilent Technology, Berkshire) as indicated by the
manufacturer. Two complementary oligonucleotides containing the desired mutation
and flanked by unmodified nucleotide sequence were designed prior mutagenesis. A
PCR mix containing the DNA template, the forward and reverse primers, dNTP
mixture, 10X reaction buffer and the PfuTurbo DNA polymerase was placed in a
thermocycler for the amplification of mutated DNA. One microliter of Dpn I restriction
enzyme was then added directly to the amplification reaction and incubated 1 h at 37
ºC to digest the parental non mutated DNA. Next, the sample reaction was
transformed into XL1-Blue competent cells, as described in the above section, and
DNA was extracted using the miniprep kit to determine whether the DNA contains the
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desired mutation. All plasmids DNA were sequenced at the Medical research Council
DNA Core laboratory using an ABI 7500 PRISM automated sequencer.
2.7.3 Plasmid DNA transfection
Cells were seeded into 10 cm dish to reach confluency approximately 60%.
Plasmids DNA (Table 2.6) were transfected using Fugene 6 (Roche Applied
Sciences, UK). The ratio 3:1 (µl of Fugene 6: µg of DNA) was used as recommended
by the manufacturer.
For one 10 cm dish, 988 µl of DMEM (containing no supplement) was added to a
sterile eppendorf tube and 12 µl of Fugene 6 was added carefully. The mixture was
shaken and left at RT for 5 min. 3 µg of plasmid DNA (Table 2.6) was added, mixed
and left at RT for 15 min. the mixture was then added gently to the dish containing
cells and 10 ml medium. Overexpressions of desired proteins were confirmed 24 h
after transfection after protein extraction and western blotting.
Vector Selection From/reference
pcDNA3-FOXM1 Ampicillin Our laboratory
pcDNA3-ΔN-FOXM1 Ampicillin Our laboratory
HEG0 (ERα) Ampicillin Tora et al, 1989, EMBO
pcDNA3-Flag- ERβ Ampicillin J. Hartman
pcDNA3-Flag-p53 Ampicillin N. Hadjji
pCMV-E2F-1 Ampicillin Helin et al, 1993
pFlag-Nbs1 Ampicillin Kou-Juey Wu (Wu et al, 2007)
Table 2.6 Expression vectors
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2.7.4 Luciferase assay
For promoter analysis, cells were treated with indicated drugs for Firefly/Renilla
luciferase assays using the Steadyliteplus reporter assay system (Perkin Elmer,
Cambridgeshire, UK) according to manufacturer’s instructions. Briefly, the substrate
(luciferin, ATP, magnesium and molecular oxygen) was added to the cells (seeded in
96-well plate) and the luminescence was measured after 15 min using a microplate
reader (BMG Labtech, Offenburg, Germany). Subsequently, the substrate
(coelenterate luciferin and molecular oxygen) for the Renilla was added to the
previous mix and the cells, and luminescence was measured after 15 min. The ratio
of luminescence of luciferase Reporter/Renilla reporter was calculated. All
experiments were performed in triplicate.
Cells were transfected with the promoter constructs (Table 2.7) and 5 ng of
Renilla (pRL-TK; Promega, Southampton, UK) as internal transfection control using
Fugene-6 according to manufacturer’s instructions (Roche Diagnostics Ltd, Burgess
Hill, UK). For some experiments, cells were transfected with human FOXM1 or NBS1
promoters and 5 ng of Renilla alone, or in combination with 10 ng and 30 ng of
expression vectors.
Promoter constructs Length From
FOXM1 promoter
pGL3-Full length 2.4kb René H. Medema
pGL3-Hind III 1.4kb René H. Medema
pGL3-ApaI 296bp René H. Medema
pGL3-ApaI-E2Fmut1 296bp Made in the laboratory
pGL3-ApaI-E2Fmut2 296bp Made in the laboratory
pGL3-ApaI-E2Fmut1/2 296bp Made in the laboratory
NBS1 promoter
pXP2-NBS1-FHK 1500bp Kou-Juey Wu
pXP2-NBS1-FHKmut 1500bp Made by myself
Table 2.7 Promoter constructs
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2.7.5 Host cell reactivation assay (HCR)
The HCR assay is an assay measuring the DNA repair of single strand damaged
DNA introduced into cells. This assay was performed using a damaged luciferase
reporter vector, in which a CMV promoter drives the transcription of the luciferase
gene. The plasmid harbouring luciferase was damaged prior transfection by a nicking
endonuclease Nb.Bts1 (New England Biolabs) and was repaired by the cellular DNA
repair machinery and only fully repaired plasmid will transcribe correctly to generate
active luciferase.
Two micrograms of the cyclinB1 promoter luciferase reporter plasmid was
damaged using 10,000 units/ml of a nicking endonuclease Nb.BtsI for 2 h at 37 ºC
(R0707S, New England Biolabs Ldt, Herts, UK) and 40 ng of plasmids were
transfected along with 5 ng of Renilla plasmid (pRL-TK; Promega, Southampton,
UK). One unit is defined as the amount of enzyme required to convert 1 µg of
supercoiled plasmid to open circular form in 1 hour at 37°C in a total reaction volume
of 50 µl. Each digestion was run on 1% agarose gel to confirm that the plasmid was
linearized.
The endonuclease Nb.Bts1 cleaves DNA as a heterodimer of one large subunit
(B subunit) and one small subunit (A subunit); and, in the absence of their small
subunits, the large subunits behave as sequence-specific DNA nicking enzymes and
only nick the bottom strand of the sequences at this position:
After the indicated time points, luciferase activity was measured and normalised
against the internal control Renilla. Because of the variation in the transfection
efficiency between undamaged and damaged plasmids, the percentage of luciferase
recovery was determined comparing to the first time point at 0 h.
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Figure 2.2 Host Cell Reactivation. Damaged pGL3-cyclinB1 luciferase reporter plasmid is transfected transfected along with undamaged Renilla plasmid. Luciferase activity was measured and normalised against the internal control Renilla.
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2.8 RNA INTERFERENCE
Transient RNA interference was used to specifically repress the expression of
chosen genes and performed using small-interfering RNA (siRNA).
In this study, cells were transfected with the SMARTpool siRNAs purchased from
Dharmacon (Lafayette, CO) using OligofectAMINE (Invitrogen, Paisley, UK)
according to manufacturer’s instructions. Briefly, cells were seeded in a 6-well plate
24 h prior transfection to reach a confluency of 50 % and the specific target gene was
silenced using 50 nM of siRNA. For each well, 70µl of Optimem was mixed with 5 µl
of oligofectAMINE (Invitrogen, Paisley, UK) and incubated at RT for 10 min. This
solution was then combined with 250 µl Optimem and 7,5 µl siRNA oligos for each
gene. After 25 min incubation, 160 µl Optimem was added to the mixture reaching a
final volume of 500 µl, which was added to the cells previously washed with 1X PBS.
The 6-well plates were incubated at 37 °C for 4 h and 2 ml of culture medium
containing 10 % FCS was subsequently added to cells to prevent toxic effects. Cells
were harvested 24 h after transfection for protein and RNA analysis. SMARTpool
FOXM1 siRNA (M-009762-00), ERα siRNA (L-003401-00), p53 siRNA (L-003329-
00), p21Cip1 (L-003471-00), ATM (L-003201-00), NBS1 (L-009641-00), CHK1 (L-
003255-00), CHK2 (L-003256-000) and siCONTROL non-targeting siRNA were used.
All experiments were performed with a control mock condition. As control mock
conditions showed the same results as the non/targeting siRNA, control mock
condition was not shown in the data.
2.8 IMMUNOFLUORESCENCE MICROSCOPY
10000 cells per well were seeded into 8-well culture slide chambers (BD
Falcon™, Oxford, UK) for confocal microscopy and 5000 cells per well into 96-well
plate, black-walled with clear bottom (BD Falcon™, Oxford, UK) for foci/staining
quantification with Image Xpress (Molecular Devices, Berkshire, UK). After 24 h, cells
were treated with epirubicin 1 µmol/L for indicated times and then fixed and
permeabilized with 100 % methanol for 10 min at RT. Wells were washed three times
with 1X PBS and blocked with 5 % goat serum for 1 h at RT. Then, fixed cells were
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incubated with a specific primary antibody for FOXM1 (Santa Cruz, sc-500) (1:250),
P-ATM (Ser1981) (1:120) (Upstate, MAB3806), P-CHK2 (Thr68) (C13C1) or for P-
H2AX (Ser139) (Upstate, JBW301) (dilution 1:250) overnight at 4 ºC, followed by
Alexa 488 (green)-conjugated anti-secondary antibody or Alexa 555 (red)-conjugated
anti-secondary antibody (Invitrogen, Molecular Devices, UK) for 1 h at RT. Cells were
counterstained with TO-PRO®-3 iodide or DAPI (Invitrogen, Molecular Devices, UK)
to show the nuclei. Specific staining was visualised and images captured with Zeiss
LSM 500 system confocal microscope. Foci quantification was performed with Image
Xpress system microscopy and analysed with MetaMorph software (Molecular
Devices, Berkshire, UK).
2.9 SRB assay
The cell survival was determined using the sulforhodamine B (SRB) colorimetric
assay previously described (Skehan, 1990). The SRB dye binds proteins of the cell. It
is an anionic aminoxanthene dye that forms an electrostatic complex with the basic
amino acid residues of proteins under moderately acid conditions, which provides a
sensitive linear response. Because the binding is stoichiometric, the quantity of dye
dissolved from the stained and fixed cells is proportional to cell mass and
representative of cell density. Therefore, the SRB assay detects the number of cells
but not cell proliferation. Approximately 5000 cells per well seeded in 96-well plates
were fixed with 100 µl of cold 40 % trichloroacetic acid for 1 h at 4 °C. After three
washes with cold water, cells were stained with 0.4 % sulforhodamine B (that binds to
protein basic amino acid residues) for 1 h at RT. Cells were then rinsed three times
with 1 % acetic acid and left to dry overnight. The protein-bound dye was dissolved
the next day in 10 mM Tris base solution for 30 min and measured at 495 nm using a
Sunrise™ plate reader (Tecan Group Ltd). The results obtained were expressed as
the mean of eight replicates relative to the results obtained for the vehicle control at 0
h providing the percentage of cell survival.
This assay is widely used for in vitro cytoxicity screening. This assay has been
used for high-throughput drug screening at the National Cancer Institute. Studies
undertaken by groups showed that results from the SRB assay correlates well with
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the MTT assay (tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) (Vichai and Kirtikara 2006).
Controls: all SRB assays with siRNA condition were performed with a mock
control. No difference has been found compared with the non-targeting siRNA,
therefore the mock data have not been included for clarity purpose.
2.10 CELL CYCLE ANALYSIS
Cell cycle analysis was performed using propidium iodide (PI) staining alone.
Cells were trypsinized, collected by centrifugation, and washed in 1X PBS before
fixing in 70 % ethanol. Fixed cells were washed twice in 90 % ethanol and then re-
suspended in 1X PBS containing propidium iodide (1 mg/ml) supplemented with
RNase (20 units/ml) for 15 min at 4 °C. The single cell suspensions were analysed on
a FACSCalibur flow cytometer (BD Biosciences Immunocytometry Systems, San
Jose, CA) with CellQuest (BD Biosciences) acquisition software. For protein staining,
cells were rinsed in PBS 1X before fixing in 70 % ethanol overnight at 4 °C. The fixed
cells were then washed twice with 1X PBS and resuspended in 1X PBS containing
0.5 % of BSA. After centrifugation, the pellet was resuspended in 100 µl 1X PBS
containing 1 % BSA and an antibody against P-ATM (Ser1981) (1:800), P-H2AX
(Ser139) (1:400) or FOXM1-C20 (1:400) and incubated for 2 h at RT. After
centrigutation, pellets were washed in 100 µl 1X PBS containing an anti-mouse or
anti-rabbit Alexa 488 (green)-conjugated secondary antibody (1:400) (Invitrogen,
Molecular Devices, UK) for 1 h at RT in the dark. After washing, pellets were
counterstained with propidium iodide (1 mg/ml) supplemented with RNase (20
units/ml) for 15 min at RT. The samples were analysed by flow cytometry as
described above. The experiments were performed in triplicate.
2.11 STATISTICAL ANALYSIS
Statistical analyses were performed using GraphPad Prism v5.0. The statistical
significance of differences between the means of two groups was evaluated by
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paired Student’s t test and considered significant when n.s non significant, * P≤0.1,
**P≤0.01 and *** P≤0.001.
.
CHAPTER 3 FOXM1 is a transcriptional target of ERalpha and has a
critical role in breast cancer endocrine sensitivity and resistance
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3.1 Introduction
Breast cancer is the most common cancer in women in the UK. The forkhead
box family member FOXM1 was previously reported elevated in breast cancer
adenocarcinoma as well as carcinoma from other origins (Pilarsky, Wenzig et al.
2004, Kalin, Wang et al. 2006, Kim, Ackerson et al. 2006, Yoshida, Wang et al.
2007). FOXM1 is highly expressed in embryo and proliferating adult tissues, while its
expression is low in quiescent cells (Korver, Roose et al. 1997). FOXM1 regulates
the expression of cell cycle regulatory genes involved in the G1/S and G2/M phase
transitions (Laoukili, Kooistra et al. 2005). A cDNA microarray study previously
identified FOXM1 as one of the 344 ERα responsive genes in breast cancer cells
(Cicatiello, Scafoglio et al. 2004). Moreover, a recent study in our laboratory showed
a positive correlation between ERα and FOXM1 protein expression in breast cancer
cells (Madureira, Varshochi et al. 2006).
Oestrogens are the most important regulators of breast cancer growth and act
through the oestrogen receptors, ERα and ERß. ERα activity induces breast cancer
cell proliferation, while ERß is an anti-proliferative factor (Paruthiyil, Parmar et al.
2004). Oestrogens are heavily implicated in breast cancer because of their role in
stimulating breast cell division, their activity during the critical periods of breast
growth and development, and their effect on other hormones that stimulate breast
cell division. Although ERα oncogenic potential, its expression in breast cancer
patients is a good prognostic. Anti-oestrogen or endocrine therapies are effective
only in patients with ERα positive breast cancer. Clinically, tamoxifen has been the
most commonly used endocrine agent, and is suitable for both pre- and post-
menopausal women. Tamoxifen is a selective ERα modulator that blocks the binding
of oestrogen on ERα and induces the recruitment of co-repressors preventing ERα
transcriptional activity.
Treatment of breast cancer with anti-oestrogens results in G1 cell cycle arrest
and in some cases, cell death (Lykkesfeldt, Larsen et al. 1986). However, about half
of the patients who initially respond to endocrine therapy become resistant despite
continued expression of ERα (Ali and Coombes 2002, Goss, Muss et al. 2008,
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Yamashita 2008). Indeed, loss of ERα expression occurs in a minority of resistant
breast cancer while various mechanisms of endocrine resistance in ERα positive
breast cancer have been reported. ERα is regulated through transcriptional, post-
transcriptional and post-translational mechanisms, and deregulation of one of these
processes can result in endocrine resistance (Musgrove and Sutherland 2009).
Emerging data indicate that altered expression of several growth factors receptors
and overexpression of ERα co-activators can constitutively phosphorylate and
thereby activate ERα conferring breast cancer endocrine resistance.
The previous observation in our laboratory that ERα positive breast cancer cells
express higher levels of FOXM1 led to hypothesise that FOXM1 may be regulated by
ERα. Based on the recent evidence linking FOXM1 with drug resistance, I
investigated the regulation of FOXM1 by ERα and its ligands in MCF-7 cells, and
FOXM1 potential role in breast cancer endocrine resistance.
3.2 Results
3.2.1 Transcriptional regulation of FOXM1 by ERα in endocrine sensitive breast cancer cells
3.2.1.1 ERα ligands and ERα silencing modulate FOXM1 expression
To investigate whether FOXM1 is a target of ERα, the ERα positive MCF-7
breast cancer cell line and ERα negative MDA-MB-231 breast cancer cell line were
treated with ERα ligands: estradiol (E2), tamoxifen (OHT) and fulvestrant (ICI). The
efficacy of these treatments was verified by pS2 protein expression, a previously
described oestrogen responsive gene (Soulez and Parker 2001). As expected, pS2
protein expression increased with the addition of E2 and decreased upon both anti-
oestrogens OHT and ICI in MCF-7 cells (Fig. 3.1A). I further observed that E2
enhanced FOXM1 protein and mRNA expression within 24 h and remained high until
48 h in the ERα positive MCF-7 cells. By contrast, E2 did not change FOXM1 protein
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and mRNA levels in the ERα negative MDA-MB-231 cells (Fig. 3.1B). The treatments
with OHT and ICI reduced the expression of FOXM1 protein and mRNA in MCF-7
cells 24 h and 48 h after treatment, while FOXM1 expression was not affected by
these treatments in MDA-MB-231 cells (Fig. 3.1B). Notably, E2 treatment decreased
ERα protein expression in the ERα positive MCF-7 cells. Indeed, it has been reported
that E2 increases ERα turnover (Nawaz, Lonard et al. 1999, Wijayaratne and
McDonnell 2001). Moreover, ICI caused ERα degradation in the ERα positive MCF-7
cells (Long and Nephew 2006). These data have been confirmed in the ERα positive
ZR-75-1 breast cancer cell line in our laboratory (data not shown; provided by
Demetra Constantinidou).
In addition, the ability of OHT and ICI to antagonize E2-regulated FOXM1
upregulation was examined Figure 3.2. Therefore MCF-7 cells were stimulated by E2
for 4 h and then treated with OHT or ICI from 0 h to 48 h. The result showed that the
effects of OHT and ICI following E2 stimulation were similar to those observed under
normal growth conditions (Fig. 3.2). The ERα targets, pS2 and cyclin D1, and
FOXM1 targets, PLK and CDC25B, were induced by E2 and repressed by the
addition of OHT and ICI. Notably, FOXM1 expression pattern varied between Figures
3.1 and 3.2. Studies showed that if starvation is not perfect, some leakage might
have occurred and cells slowly accumulate material leading to S phase initiation even
during the period of incubation in low serum. Cells closer to initiation (later in S
phase) can reach initiation mass sooner than cells earlier in the G1 phase. Cells later
in the G1 phase at the time growth arrest are less likely to have a delayed cell
division. Cells earlier in the cycle will not accrue enough leakage to initiate DNA
synthesis and thus will exhibit a delayed cell division. FOXM1 expression pattern
varies throughout the cell cycle. Therefore, it is possible that starvation was not
reached Figure 3.2 leading to cells in different cell cycle phases with different FOXM1
protein expression levels (Cooper 2003, Cooper 2003).
In conclusion, the expression of FOXM1 protein and mRNA is upregulated by
the ERα agonist and downregulated by ERα antagonists only in the ERα positive
MCF-7 cells suggesting that ERα regulates FOXM1 expression. To provide further
evidence of FOXM1 regulation by ERα, the effect of ERα silencing on FOXM1 protein
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and mRNA levels was studied in MCF-7 cells. After 24 h transfection, ERα siRNA
effectively silenced ERα at protein level and mRNA levels, and reduced the levels of
FOXM1 protein and mRNA significantly (Fig. 3.3). In accordance with previous
findings in the laboratory, FOXM1 siRNA also reduced ERα protein and mRNA levels
(Madureira, Varshochi et al. 2006). Unfortunately, the quality of the western blot for
this experiment is very poor and should be repeated.
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Figure 3.1 Expression of FOXM1 and ERα in response to E2, tamoxifen and ICI treatments in breast cancer cell lines. MCF-7 (A) and MDA-MB-231 (B) cells were cultured in 5 % double-charcoal striped FCS and phenol red free medium for 24 h before being stimulated with 10-8 mol/L of E2. Breast cancer cells cultured in 10 % FCS and phenol red medium were also treated with 10-6 mol/L of OHT or 10-7 mol/L of ICI. At times indicated, cells were collected and analysed for FOXM1, ERα, pS2 and ß-tubulin expression by western blotting. FOXM1 mRNA levels of these cells were also analysed by RT-qPCR, and normalized with L19 RNA expression. All data shown represent the averages of data from three independent experiments, and the error bars show the standard deviations.
89
Figure 3.2 Induction of FOXM1 expression by E2 is antagonized by OHT and ICI in MCF-7 cells. MCF-7 cells were cultured in 5 % double-charcoal striped FCS and phenol red free medium for 24 h before stimulation with 10-8 mol/L of E2 (-4h). Four hours after E2 stimulation, the MCF-7 cells were treated with 10-6 mol/L of OHT or 10-7 mol/L of ICI for the indicated times. Cells were collected and analysed for FOXM1, ERα, PLK, CDC25b, CYCLIN D1 and ß-tubulin protein expression using western blotting.
90
Figure 3.3 Effects of ERα silencing on the expression of FOXM1. MCF-7 cells were transiently transfected with ERα, FOXM1 or NS (non-specific) siRNA (100 nmol/L) with oligofectAMINE according to the manufacturer instructions. After 24 h transfection, cells were analysed for protein levels by western blot (A.) using specific antibodies as indicated and for mRNA levels of FOXM1 (B.) and ERα (C.) by RT-qPCR. All data shown represent the averages of three independent experiments, and the error bars show the standard deviations. Statistical analyses were done using Student’s t test. *, P≤0.1, **, P≤0.01 and ***, P≤0.001, significant and n.s, non significant.
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3.2.1.2 FOXM1 promoter responds to ERα ligands
The RT-qPCR data obtained in Figure 3.3 showed that FOXM1 is
transcriptionally regulated by ERα. In order to elucidate whether FOXM1 is regulated
at promoter level by ERα, Demetra Constantinidou and myself transiently co-
transfected an ERα expression vector with three different FOXM1 promoter
constructs in a luciferase reporter vector (Fig. 3.4A), previously made by Demetra
Constantinidou in our laboratory, in COS-1 cells. We observed a 2-fold increase of
luciferase activity of the 2 kb full length FOXM1 (pGL3-Full-length) and 1.5-fold
increase of the luciferase activity of the 1.3 kb Hind III truncation constructs (pGL3-
Hind III) 24 h after E2 treatment (Fig. 3.4B). The highest luciferase activity was
obtained with the 300 pb Apa I truncation construct (pGL3-Apa I) that showed an
enhancement of 2.5-fold upon 4 h E2 treatment, consistent with a previous study
identifying a similar region of the FOXM1 promoter that responds to serum
stimulation (Korver, Roose et al. 1997). As positive control, we performed this
experiment with the pS2 promoter (pGL3-ERE) under the luciferase reporter gene
and observed an increase of 5-fold of the luciferase activity following E2 treatment.
The addition of OHT for 24 h following E2 treatment resulted in a repression of all
luciferase reporter gene activities previously induced by E2 (Fig. 3.4B).
These reporter assays were performed with the help of Demetra
Constantinidou, who examined the proximal FOXM1 promoter sequence and
identified potential ERα-responsive elements (EREs) by using the Transcription
Element Search System (TESS website). These analyses revealed an ERE-like
element located at -45 pb from the transcription start site that could explain the
responsiveness to ERα ligands of the three FOXM1 promoters constructs used.
Consequently, studies were focused on the characterization of the ERE-like element
at -45 pb in the Apa I promoter fragment. By introducing mutations in both arms of
the ERE-like palindrome of the Apa I truncation (Bourdeau, Deschenes et al. 2004)
(Fig. S.D.7.1), Demetra Constantinidou observed by luciferase assay that the ERE3
mutant (mERE3) lost the majority of the responsiveness to E2 (Fig. S.D.7.2).
92
Figure 3.4 ERα induces the transcriptional activity of the human minimal FOXM1 construction gene. Effect treatment with E2 alone or in combination with OHT and transient expression of ERα on FOXM1 promoter activity. A. Schematic representation of the 2.4 kb full length, 1.4 kb Hind III and 0.3 kb Apa I FOXM1-luciferase reporter constructs previously made in the laboratory. B. COS-1 cells were cultured in 5 % double-charcoal striped FCS and phenol red free medium were transiently transfected with 20 ng of either the empty pGL3-basic, or FOXM1 truncations: pGL3-Full length, pGL3-Hind III, pGL3-ApaI, or the control pGL3-ERE (pS2) promoter and 0 ng or 10 ng of ERα expression vector in the presence or absence of 10-8 mol/L of E2 alone for 4 h or 10-8 mol/L of E2 for 4 h followed by 10-6 mol/L of OHT for 24 h. Cells were treated 24 h after transfection and harvested for luciferase assay. All relative luciferase activity values were corrected by co-transfected Renilla activity. All data shown represent the averages of data from three independent experiments, and the error bars show the standard deviations.
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3.2.1.3 ERα and HDAC2 bind on the ERE-like site of FOXM1 promoter in vitro
To investigate whether ERα binds to the previously characterized ERE-like site
in vitro, a pull-down assay with biotin-labeled oligonucleotides was performed using
ERα positive MCF-7 and ZR-75-1 breast cancer cell lines. Streptavidin agarose
beads were used to bind the wild-type oestrogen response element biotin-
oligonucleotide (WT ERE-biotin) after incubation with nuclear protein extracts of
breast cancer cells. Furthermore, a competition of the binding of the nuclear extract
to the WT ERE biotin-oligonucleotide by addition of an excess of either wild-type (WT
ERE-nonbiotin) or mutated (mut ERE3-nonbiotin) unlabelled-oligonucleotides was
performed (Fig. 3.5). The binding of ERα to biotinylated oligonucleotides was
analyzed by western blot and immunoblotted with an anti-ERα antibody. In Figure
3.5, I observed that ERα binding was completely abolished by the addition of
unlabelled WT ERE oligonucleotide (WT ERE-nonbiotin, lane 2), while it was only
slightly decreased with unlabeled mERE3 oligonucleotides (mERE3-nonbiotin, lane
3) under control vehicle condition in MCF-7 cells and ZR-75-1 cells. This result
indicates that the WT ERE unlabeled-oligonucleotide has a higher affinity for ERα
binding than mERE3. Moreover the effect of OHT, ICI and E2 on ERα binding on
ERE sequence was verified in MCF-7 and ZR-75-1 cells (Fig. 3.5). The results show
that ERα binding decreased in both cell lines upon ICI treatment (lane 12), whereas
ERα binding seemed to be steady following OHT (lane 6) and E2 (lane 18)
treatments. In agreement with previous studies and Figure 3.1, ICI decreased ERα
protein level, while OHT stabilized it.
Collectively, these results demonstrated that ERα binds specifically to the ERE-
like element found on FOXM1 promoter in vitro under E2 and OHT treatments.
However, these data did not explain the differential mechanism of FOXM1 regulation
by ERα upon OHT and E2, since ERα binds to the WT ERE sequence in both OHT
and E2 conditions. To address this point, pull-down assays were performed in MCF-7
cells and immunoblotted with HDAC2 antibody, a histone deacetylase specifically
overexpressed in breast cancer cells (Fig. 3.5). Interestingly, HDAC2 binds only to
94
the WT ERE biotin-oligonucleotides (WT ERE-biotin, lane 4 and 6) upon OHT but not
under E2 (lane 16 and18) conditions. This result suggests that OHT treatment
induces ERα co-repressors binding including HDAC2 to repress FOXM1 expression.
Surprisingly, HDAC2 was detected upon ICI treatment on the WT ERE biotin-
oligonucleotides (WT ERE-biotin, lane 10). This result raised two issues for this
experiment: the lack of a control input and the lack of protein extract quantification
after incubation with the streptavidin-beads. These controls could add a quantitative
value to the experiment and determine whether HDAC2 binds ERE under ICI
treatment.
Figure 3.5 ERα binds directly to the ERE-like site on FOXM1 promoter in vitro. MCF-7 and ZR-75-1 cells were cultured in 5 % double-charcoal striped FCS and phenol red free medium for 24 h before being stimulated with 10-8 mol/L of E2 for 24 h. Cells cultured in 10 % FCS and phenol red medium were treated with 10-6 mol/L of OHT, 10-7 mol/L of ICI or control vehicle (control) for 24 h. Nuclear extracts from MCF-7 and ZR-75-1 cells were incubated with biotin-oligonucleotides representing region of the FOXM1 promoter containing the wild type ERE (WT ERE-biotin) in the absence or presence of molar excess of non-biotinylated ERE3 (mut ERE3-nonbiotin) or wild type ERE (WT ERE-nonbiotin) oligonucleotides. Proteins binding to the biotinylated oligonucleotides were pulled-down using streptavidine agarose beads and analysed by western blot using ERα antibody for ZR-75-1 cells, and with ERα and HDAC2 antibodies for MCF-7 cells. Nuclear extracts from MCF-7 cells were incubated with biotin-oligonucleotides representing region of the FOXM1 promoter containing the wild type ERE or the mutated ERE3 site in the absence or presence of molar excess of non-biotinylated ERE3 or wild type ERE oligonucleotides. Proteins binding to the biotinylated oligonucleotides were pulled-down using streptavidine agarose beads and analysed by western blot using HDAC2 antibody.
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3.2.1.4 ERα binds specifically to FOXM1 promoter in vivo
I further studied the in vivo binding of ERα on FOXM1 promoter in the MCF-7
and ZR-75-1 cell lines after OHT or ICI treatments using a semi-quantitative
chromatin immunoprecipitation (Fig. 3.6A). The ERα-bound DNA was amplified with
the FOXM1 ERE site primers. In agreement with the pull-down studies, ERα
occupied FOXM1 promoter in control vehicle conditions and its occupancy decreased
clearly after ICI treatment and was not affected by OHT treatment. For negative
controls, samples were IP with non-specific IgG antibodies and PCR was performed
on a region where ERα is absent. Semi-quantitative ChIP assays also showed an
increase in the recruitment of HDAC1 and HDAC2 upon OHT treatment. Consistent
with this finding, I showed a decrease of acetylated H3 and H4 on FOXM1 promoter,
indicating that OHT treatment caused the recruitment of HDACs that confer
transcriptional repression to the ERE region of FOXM1 promoter (Fig. 3.6B).
Histones H3 and H4 are ubiquitously expressed and enriched on actively transcribed
region. Therefore, this experiment should be repeated with appropriate negative
controls including histone deacetylases such as HDACs or SIRTs.
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Figure 3.6 Chromatin immunoprecipitation (ChIP) analysis of the human FOXM1 promoter. A. MCF-7 and ZR-75-1 cells untreated or treated with 10-6 mol/L of OHT or 10-7
mol/L of ICI for 24 h were used for ChIP assays using anti-IgG control, anti-ERα antibodies as indicated. B. MCF-7 untreated or treated with 10-6 mol/L of OHT for 24 h were used for ChIP assays using anti-IgG control and antibodies against acetylated H3 and H4, HDAC1 and HDAC2 as described above. After crosslink reversal, the co-immunoprecipitated DNA was amplified by PCR using primers amplifying the FOXM1 ERE containing region (−184/+4) and a control region (−1157/−1257), and resolved on 2 % agarose gel. Representative data from three independent experiments are shown.
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3.2.1.5 FOXM1 silencing is cytotoxic for MCF-7 cells independent of the E2 mitogenic effect
Since the previous results showed FOXM1 as an ERα-responsive gene, I
investigated the effects of FOXM1 knockdown on the survival of MCF-7 cells.
Western blot analysis Figure 3.7A showed that FOXM1 was efficiently silenced by
specific siRNA and that ERα expression was also decreased by FOXM1 siRNA. This
is in line with previous findings in our laboratory that FOXM1 regulates ERα
expression (Madureira, Varshochi et al. 2006). MCF-7 cells were oestrogen-starved
for 48 h and then stimulated with E2 in the presence or absence of FOXM1 siRNA
(Fig. 3.7). Notably, E2 treatment decreased ERα protein expression as already
observed in Figure 3.1. The results of SRB assay showed that FOXM1 silencing
induced the number of cells in the ERα positive MCF-7 cells following E2 or control
vehicle (Fig. 3.7B). Taken together, the results indicate that FOXM1 has a role in the
survival of ERα positive MCF-7 cells independent of E2. It would also be a good
control to repeat this experiment with siRNA against ERα and FOXM1 combined to
confirm that FOXM1 overcome the role of ERα in cell proliferation of MCF-7 cells.
The effect of FOXM1 silencing on MCF-7 cell proliferation should be studied using
different methods: vital staining or measure of DNA synthesis. Trypan blue selectively
colour dead cells in blue, while the quantification of 3H-thymidine or
bromodeoxyuridine incorporated into newly synthetized DNA indicate proliferation.
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Figure 3.7 Effects of FOXM1 silencing on E2-induced proliferation of MCF-7 cells. MCF-7 cells were transiently transfected with non-specific (NS) and specific siRNA against FOXM1 (FOXM1) (100nmol/L) using oligofectAMINE according to the manufacturer instructions and, starved for 48 h and incubated with 10-8 mol/L of E2 for 24 h and analysed by western blotting with anti-FOXM1, ERα, β-tubulin (A.). Treatment with 10-8 mol/L of E2 for 0, 24, 48 h was performed before cells were harvested for SRB assays (B.). Representative data from three independent experiments are shown.
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3.2.2 Deregulation of FOXM1 in tamoxifen resistant breast cancer cells
3.2.2.1 Deregulation of FOXM1 protein and mRNA expression in tamoxifen resistant cells
Since the majority of the patients who develop resistance to hormonal therapy
are ERα positive, a dysfunction of ERα could result in FOXM1 deregulation and
endocrine resistance in breast cancer cells. To address this question, I studied
FOXM1 expression in tamoxifen sensitive MCF-7 cells (MCF-7) and tamoxifen
resistant MCF-7 cells (MCF-7TAMR4) after treatment with OHT. Consistent with
Figure 3.1, OHT treatment caused a drastic reduction in FOXM1 protein and mRNA
levels after 24 h until 72 h in MCF-7 cells, whereas no significant decrease was
observed in MCF-7TAMR4 cells (Fig. 3.8A). Similar to FOXM1 expression pattern,
FOXM1 target genes such as PLK, CDC25B, cyclin A and ERα showed a decrease
in their protein expression in MCF-7 cells, but did not change in MCF-7TAMR4 cells,
consistent with FOXM1 expression levels (Fig. 3.8A).
Additionally, to elucidate whether FOXM1 is involved in tamoxifen resistance,
our group developed a stable MCF-7 cell line overexpressing a constitutive active
form of FOXM1, the N-terminal deleted FOXM1 form (ΔN-FOXM1) which was
previously described (Park, Wang et al. 2008). The protein and mRNA expression of
FOXM1 under a constitutively active promoter (CMV) in MCF-7 cells led to a high and
constant FOXM1 and ΔN-FOXM1 protein expression, and constant FOXM1 mRNA
expression even following 72 h OHT treatment (Fig. 3.8B). As in MCF-7TAMR4 cells,
FOXM1 targets protein expressions are consistent with FOXM1 expression pattern
and remained unchanged. Moreover, to confirm whether FOXM1 has a pivotal role in
tamoxifen resistance, different clones of MCF-7 cells (Pool of all clones, Clone 1,
Clone 3, Clone 4) stably transfected with the full length wild-type FOXM1 (MCF-7-
FOXM1) previously generated in the laboratory were used (Fig. 3.8C). Western blot
analysis showed that the transfection of the full length FOXM1 abolished the
downregulation of FOXM1 in clones 2 and 3, while FOXM1 was decreased in clone 1
and pool following OHT. This suggests that FOXM1 might be regulated at post-
transcriptional levels by tamoxifen as FOXM1 expression is driven by CMV promoter
in this experiment. Taken together, the full length FOXM1 overexpression partially
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prevented the downregulation of FOXM1 targets including ERα, CDC25b, PLK and
CYCLIN B1 in response to tamoxifen treatment (Fig. 3.8C).
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Figure 3.8 Full length and partial FOXM1 overexpression reduced the downregulation of FOXM1 and its target genes following tamoxifen treatment. MCF-7, MCF-7TAMR4 and MCF-7 ∆N-FOXM1 cells were treated with 10-6 mol/L of OHT in a time course of 72 h. A. Cell lysates were prepared at the times indicated, and the expression of FOXM1, ERα, CDC25B, PLK, and β-tubulin were analysed by Western blotting. B. Cells were harvested and FOXM1 mRNA levels were analysed by RT-qPCR, normalised with L19 housekeeping gene. C. Wild-type MCF-7 and overexpressing-FOXM1 MCF-7 (pool of clones, clone 1, 2 and 3) cell lysates were prepared at the times indicated, and the expression of FOXM1, ERα, PLK, CDC25B, CYCLIN B1, CYCLIN D1 and β-tubulin were analyzed by Western blotting.
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3.2.2.2 Reduced G1 cell cycle arrest in tamoxifen resistant cells after OHT
I performed cell cycle analysis of the MCF-7, MCF-7TAMR4 and ΔN-FOXM1
MCF-7 cells following treatment with OHT (Fig. 3.9A). The results showed that OHT
caused predominantly a G1 cell cycle arrest in the parental MCF-7 cells, but had
comparatively lower effects on the cell cycle distribution of the MCF-7TAMR4 and ΔN-
FOXM1 MCF-7 cells (Fig. 3.9B). The calculation of the percentage of increase
relative to 0h in cells in G1 phase showed that OHT caused a G1 cell cycle arrest in
MCF-7TAMR4 at a lower extend than in the wild-type MCF-7 cells, whereas OHT did
not increase the number of cells in G1 phase in ΔN-FOXM1 MCF-7 cells (Fig.3.9B).
The cell cycle profile of the MCF-7 cells stably transfected with the wild-type
FOXM1 (MCF-7-FOXM1) demonstrated that these cells underwent a cell cycle arrest
at G1, concomitant with FOXM1 downregulation, but at a lower extend compared
with MCF-7 cells transfected with the empty vector (Fig. 3.10). Similar to the western
blot, pool and clone 1 MCF-7 cells did not show a strong difference in the number of
cells in G1 phase compared with the wild-type MCF-7 cells, while clones 2 and 3
showed a lower increase in cells in G1 phase after 72h treatment. Collectively, these
results indicate that FOXM1 has a role in mediating the G1 cell cycle arrest OHT-
induced, and that its overexpression reverses MCF-7 sensitivity to G1 cell cycle
arrest OHT-induced. Furthermore it would be interesting to overexpress a
transcriptionally dead FOXM1 to demonstrate that FOXM1 activity is required for the
inhibition of the G1 cell cycle arrest OHT-induced. At this point, it would also have
been interesting to compare the proliferation of MCF-7, MCF-7TAMR4 and ΔN-
FOXM1 MCF-7 cells following OHT.
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Figure 3.9 Cell cycle regulation in wild-type (MCF-7), tamoxifen resistant (MCF-7TAMR4) and constitutively active ∆N-FOXM1 expressing MCF-7 cells in response to tamoxifen treatment. MCF-7, MCF-7 TAMR4 and MCF-7 ∆N-FOXM1 cells were treated with 10-6 mol/L of OHT in a time course of 72 h. A. Cell cycle phase distribution was analyzed by flow cytometry after propidium iodide staining. Percentage of cells in each phase of the cell cycle (sub-G1, G1, S, and G2/M) is indicated. Representative data from three independent experiments are shown. B. Percentage increase in cells in G1 phase relative to 0h. Statistical analyses were done using Student’s t test. *, P≤0.1, **, P≤0.01 and ***, P≤0.001, significant and n.s, non significant.
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Figure 3.10 FOXM1 upregulation rescues OHT-induced cell growth arrest and decrease of endogenous FOXM1 in response to tamoxifen treatment. A. Wild-type MCF-7 and overexpressing-FOXM1 MCF-7 cells (pool of clones, clone1, 2 and 3) were fixed at 0, 24, 48, and 72 h after OHT treatment, and cell cycle phase distribution was analysed by flow cytometry after propidium iodide staining. Percentage of cells in each phase of the cell cycle (sub-G1, G1, S, and G2/M) is indicated. B. Percentage increase in cells in G1 phase relative to 0h. Statistical analyses were done using Student’s t test. *, P≤0.1, **, P≤0.01 and ***, P≤0.001, significant and n.s, non significant.
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3.2.2.3 Combination of OHT and FOXM1 silencing has a cytostatic effect on MCF-7 tamoxifen resistant cells
Previous results showed that the constitutively active form of FOXM1 and the
full length FOXM1 reduced the G1 cell cycle arrest induced by OHT. I next tested
whether FOXM1 silencing would affect the survival of tamoxifen resistant cells to
OHT treatment. The downregulation of FOXM1 by specific siRNA was verified by
western blot (Fig. 3.11A). In the non-specific siRNA condition, the expression of
FOXM1 was enhanced during the time course due to the fact that FOXM1 is a
proliferative factor (Fig. 3.11A). Oppositely, FOXM1 protein expression remained low
until 72 h in cells transfected with specific FOXM1 siRNA (Fig. 3.11A). Interestingly,
western blot analysis also showed a downregulation of ERα protein expression upon
FOXM1 silencing indicating that ERα is still under FOXM1 regulation in these cells.
Additionally, I performed the SRB assay and observed that FOXM1 silencing
decreased the survival of tamoxifen resistant cells (Fig. 3.11B). The combination of
FOXM1 siRNA and OHT treatment had a cytostatic effect on these cells after OHT
treatment (Fig. 3.11B). A recent publication showed that Thiostrepton directly
interacts with FOXM1 protein to reduce FOXM1 transcriptional activity (Hegde,
Sanders et al. 2011). It would be interesting to compare the effect of FOXM1
silencing to this inhibitor on the tamoxifen resistant cell proliferation and survival.
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Figure 3.11 FOXM1 silencing rescues tamoxifen anti-growth effect in MCF-7TAMR4 cells. MCF-7TAMR4 cells were transiently transfected with non-specific or FOXM1 siRNA for 24 h (100nmol/L) using oligofectAMINE according to the manufacturer instructions, and were harvested at different time after transfection and analysed by western blot using FOXM1, ERα and β-tubulin antibodies (A.) and harvested for SRB assays (B.). Statistical analyses were done using Student’s t test. *, P≤0.1, **, P≤0.01 and ***, P≤0.001, significant and n.s, non significant.
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3.2.3 Potential mechanisms of tamoxifen resistance
3.2.3.1 FOXM1 phosphorylation and transcriptional activation
Western blot analysis Figure 3.12 showed that CYCLIN A, but not its catalytic
partner CDK2, was downregulated after 24 h OHT treatment in MCF-7 cells and this
decline in CYCLIN A level occurred in a slower kinetic in MCF-7TAMR4 and ΔN-
FOXM1 cells. Consistent with this, the CDK2 activity revealed by the phosphorylation
of pRB on Threonine 821 decreased drastically in MCF-7 cells compared with MCF-
7TAMR4 and ΔN-FOXM1 cells. Given that the complex CYCLIN A/CDK2
phosphorylates FOXM1 and can activate its transcriptional activity (Wierstra and
Alves 2006, Laoukili, Alvarez et al. 2008, Laoukili, Alvarez-Fernandez et al. 2008,
Park, Wang et al. 2008), this finding suggests a potential mechanism by which
FOXM1 can cause tamoxifen resistance in MCF-7TAMR4 and ΔN-FOXM1 cells.
Western blot analysis also showed that the level of CYCLIN D1 and its
associated activity revealed by the CDK4 phospho-pRb (Ser807/811) antibody were
overexpressed in the ΔN-FOXM1 cells and maintained in the MCF-7TAMR4 cells. In
addition, the stable MCF-7-FOXM1 cell line also showed an increase in the
expression levels of CYCLIN D1, particularly in clones 2 and 3 (Figure 3.8). Given
that the complex CYCLIN D1/CDK4 phosphorylates FOXM1 in multiple sites and can
activate its transcriptional activity (Anders, Ke et al. 2011), this finding suggests
another potential mechanism by which FOXM1 can cause tamoxifen resistance in
MCF-7TAMR4 and ΔN-FOXM1 cells.
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Figure 3.12 Constitutively active ∆N-FOXM1 expressing MCF-7 cells show the same protein expression pattern as MCF-7TAMR4 cells in response to tamoxifen treatment. MCF-7, MCF-7TAMR4 and MCF-7 ∆N-FOXM1 cells were treated with 10-6 mol/L of OHT in a time course of 72 h. Cell lysates were prepared at the times indicated, and the expression of CYCLIN A, CDK2, CYCLIN D1, CDK4, P-pRB(cdk2), P-pRB(cdk4), total pRB and β-tubulin were analysed by Western blotting.
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3.2.3.2 ERα overexpression and silencing do not alter FOXM1 expression in tamoxifen resistant cells
Western blot analysis showed that ERα overexpression in MDA-MB-231 ERα-
negative cells had no effect on FOXM1 protein expression (Fig. 3.13), while ERα-
agonist increased FOXM1 protein expression in MCF-7 ERα positive cells (Fig. 3.1).
Similarly, ERα silencing did not affect FOXM1 protein expression in MCF-7 tamoxifen
resistant cells, while ERα silencing decreased FOXM1 protein level in MCF-7 cells
(Fig. 3.3). These data suggest that FOXM1 regulation is controlled by other mitogenic
factors in ERα-negative and tamoxifen resistant cells.
Figure 3.13 ERα ectopic expression and silencing in the tamoxifen resistant ERα-negative MDA-MB-231 and ERα-positive MCF-7TAMR4 breast cancer cells. MDA-MB-231 cells were transfected with increasing amounts of ERα. MCF-7TAMR4 cells were transiently transfected with non-specific siRNA and siRNA targeting ERα for 24 h (100nmol/L). The expression of FOXM1, ERα, CYCLIN D1, pS2 and β-tubulin was analysed by western blotting.
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3.2.3.3 Protein deregulations in tamoxifen resistant cells
ERα is inhibited by the binding of tamoxifen which induces the recruitment of
co-repressors, preventing ERα conformational changes and transcriptional activity.
Semi-quantitative ChIP assays Figure 3.6B showed that tamoxifen treatment of MCF-
7 cells induced the recruitment of HDACs (ERα co-repressor) decreasing the
acetylation levels of histones 3 and 4 as observed for two ERα target genes, pS2 and
C-MYC (Masiakowski, Breathnach et al. 1982, Carroll, Meyer et al. 2006). Thereby, it
is possible that recruitment of ERα co-repressors and co-activators is deregulated in
tamoxifen resistant cells leading to tamoxifen insensitivity. Protein analysis of AIB1
(co-activator) showed a high expression level in tamoxifen resistant MCF-7 cells even
following tamoxifen, while AIB1 expression level decreased in MCF-7 cells treated
(Figure 3.14).
A recent study showed C-MYB as an ERα target gene and determined the role
of C-MYB in the proliferation of ERα-positive breast cancer cells, but not in ERα-
negative cells (Drabsch, Hugo et al. 2007). While the role of C-MYB has been well
studied in cell growth and transformation, little is known about B-MYB family member.
Although a direct role of B-MYB in cancer has not been yet established, B-MYB is
found amplified in breast, liver, ovarian carcinomas and cutaneous T-cells
lymphomas (Forozan, Mahlamäki et al. 2000, Tanner, Grenman et al. 2000,
Zondervan, Wink et al. 2000, Mao, Orchard et al. 2003). B-MYB expression is
upregulated in metastasis compared to localised prostate tumours and its
overexpression is associated with poor prognosis in breast cancer patients
(Amatschek, Koenig et al. 2004). As FOXM1, B-MYB is a cell cycle-regulatory gene.
Its expression is induced at G0/S and reached a maximum level in S phase (Lam,
Bennett et al. 1995). A study showed that MYB mRNA and protein levels were not
affected by FOXM1 silencing, whereas FOXM1 mRNA and protein levels were both
reduced in a time-dependent manner after MYB silencing. These results suggest that
MYB is a transcriptional activator of FOXM1 (Lefebvre, Rajbhandari et al. 2010).
Therefore, altered B-MYB expression in breast cancer may affect FOXM1 expression
and be involved in tamoxifen resistance. Preliminary data on tamoxifen resistant
breast cancer cells confirmed that B-MYB is deregulated compared to wild-type MCF-
7 cells (Fig.3.14).
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Figure 3.14 AIB-1 and B-myb expression pattern in tamoxifen sensitive and resistant MCF-7 cells. Both cell lines were treated with OHT over 72 h. The expression of AIB-1, B-MYB and β-tubulin were analysed by western blotting.
In addition to ERα, ERβ is an oestrogen receptor encoded by a distinct gene
than oestrogen receptor alpha, but both bind with equal affinity oestrogens. ERα
promotes the proliferation of breast epithelium and cancer cells, while ERβ has anti-
proliferative and pro-apoptotic effects. A recent study in our laboratory has shown
FOXM1 has a target of ERβ1, but only in ERα positive breast cancer cells. MCF-7
cells collected 24 hours after transfection with pcDNA3 as a control or pcDNA3-Flag-
ERβ1 were subjected to semi-quantitative ChIP analysis with the use of an ERα
antibody and an anti-Flag antibody, which recognized the transfected Flag-tagged
ERβ1. The ChIP assays showed that there was an increase in ERβ1 recruitment to
the ERE region on FOXM1 promoter when ERβ1 is transfected. Concomitantly,
occupancy of ERE region by ERα was drastically reduced in MCF-7 cells with ERβ1
ectopic expression, indicating that ERβ1 expression caused the disassociation of
ERα from ERE region of the FOXM1 promoter (Fig. 3.15). Given that ERβ1 is
repressing FOXM1, it is possible that ERβ1 is downregulated in tamoxifen resistant
cells leading to constant cell proliferation and tamoxifen resistance.
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Figure 3.15 Chromatin immunoprecipitation of ERβ and ERα in MCF-7 cells. MCF-7 cells were transfected with ERβ and harvested for ChIP assays using anti-IgG, anti-Flag (recognized transfected ERβ) and anti-ERα antibodies as indicated. After crosslink reversal, the co-immunoprecipitated DNA was amplified by PCR using primers amplifying the FOXM1 ERE containing region (−184/+4) and resolved in 2% agarose gel.
3.3 Discussion
3.3.1 Regulation of ER and FOXM1 through a positive feedback loop in breast cancer cells
Colleagues in the laboratory previously studied FOXM1 mRNA expression and
its relationship to ERα mRNA level in breast cancer biopsy samples (Millour,
Constantinidou et al. 2010). After exclusion of the data with high levels of FOXM1
expression (upper 25th percentile), a statistically significant correlation between ERα
and FOXM1 mRNA expression was found. This is in agreement with a recent breast
cancer patient microarray dataset analysis indicating that high levels of FOXM1
mRNA expression (upper 25th percentile) are associated with poor prognosis in
breast cancer (Martin, Patrick et al. 2008). The discordance between ERα and
FOXM1 mRNA expression in patient samples with high FOXM1 expression probably
indicates that in these subjects FOXM1 expression is deregulated, with the control of
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FOXM1 transcription by ERα overridden by other mitogenic signals or genetic
changes.
A previous study in our laboratory also showed that the expression of ERα is
regulated by FOXM1 in breast cancer cell lines (Madureira, Varshochi et al. 2006). In
this report, I investigated the reciprocal regulation of FOXM1 expression by ERα.
Using breast carcinoma cell lines, I showed that FOXM1 protein and mRNA
expression are regulated by ER-ligands, including E2, OHT, and ICI (Fig. 3.1A). In
addition, I also found that depletion of ERα by RNA interference in MCF-7 cells leads
to the downregulation of FOXM1 expression (Fig. 3.3). Reporter gene assays
demonstrated that ERα activates FOXM1 transcription through an ERE located at -45
bp upstream of the transcriptional start site (Fig. 3.4) (Bourdeau, Deschenes et al.
2004). The direct binding of ERα to the FOXM1 promoter was confirmed in vitro by
DNA pull-down assays, and in vivo by semi-quantitative ChIP analysis (Fig. 3.5 and
3.6). Silencing of FOXM1 by RNA interference had a cytostatic effect on tamoxifen
resistant cells (Fig. 3.11). Conversely, ectopic expression of a constitutive active
FOXM1 form can abrogate the G1 cell cycle arrest mediated by OHT (Fig. 3.9).
3.3.2 Uncoupled ER and FOXM1 feedback loop regulation
in tamoxifen resistant breast cancer cells
The findings that tamoxifen represses FOXM1 expression in endocrine sensitive
but not in resistant breast carcinoma cell lines, and that FOXM1 overexpression can
abrogate G1 cell cycle arrest induced by tamoxifen, further suggested that
deregulation of FOXM1 may contribute to anti-oestrogen insensitivity. FOXM1 mRNA
levels are higher in tamoxifen resistant MCF-7 cells relative to wild-type MCF-7 cells,
suggesting that the main mechanism of FOXM1 regulation is transcriptional in these
cells (Fig. 3.8B). Moreover, the constitutive active FOXM1 form overexpressing cells,
where FOXM1 is under CMV promoter, showed a decrease in FOXM1 protein
expression following tamoxifen indicating a post-translational mechanism of
regulation too (Fig. 3.8C). The observation that there was no significant changes in
ERα or endogenous FOXM1 levels in the MCF-7 cells expressing the active FOXM1
form highlighted an important positive feedback mechanism between ERα and
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FOXM1, described here and previously (Madureira, Varshochi et al. 2006). This
makes FOXM1 a particularly critical ERα target gene in breast cancer development
and endocrine resistance, as the feedback loop will amplify the mitogenic action of
oestrogens. Furthermore, it seems that this positive feedback transcriptional
mechanism is uncoupled in ERα-negative breast cancer, as ERα overexpression did
not induce FOXM1 expression (Fig. 3.13). In addition, the positive feedback
transcriptional mechanism is also uncoupled in ERα-positive tamoxifen resistant
breast cancer cells as ERα silencing did not affect FOXM1 expression while FOXM1
silencing reduced ERα expression in tamoxifen resistant cells (Fig. 3.13). This finding
suggests that FOXM1 is a strong transcription activator in tamoxifen resistant cells.
3.3.3 Deregulated AIB1, an ERα co-factor, in tamoxifen resistant breast cancer cells
Over the last years, it has become evident that ERα activation is not only
dependent on the binding of ligands, but also depends on interaction between co-
factors and associated signaling pathways. Altered levels of ERα co-factors are
observed in tamoxifen resistant patients. AIB1, an ERα co-activator, is found
overexpressed in tamoxifen resistant breast cancer patients, and NCoR, an ERα co-
repressor, is reduced in tumours that acquired tamoxifen resistance (Lavinsky,
Jepsen et al. 1998, Osborne, Bardou et al. 2003). AIB1 protein analysis showed high
expression in tamoxifen resistant MCF-7 cells even following tamoxifen, while AIB1
expression decreased in MCF-7 cells treated (Figure 3.14). Semi-quantitative ChIP
assays demonstrated the mechanism by which tamoxifen represses FOXM1
expression in MCF-7 cells. Tamoxifen treatment of MCF-7 cells induced the
recruitment of HDACs decreasing the acetylation levels of histones 3 and 4 as
observed for two ERα target genes, pS2 and C-MYC (Masiakowski, Breathnach et al.
1982, Carroll, Meyer et al. 2006). Thereby, I speculate that recruitment of ERα co-
activators is deregulated in tamoxifen resistant cells, which might affect the regulation
of FOXM1 by ERα. However, ERα silencing did not affect FOXM1 expression in the
tamoxifen resistant cells, which indicates that FOXM1 regulation is controlled by
other mitogenic factors. For instance, the cell cycle regulator CYCLIN D1 regulates
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positively FOXM1 and its overexpression is associated with tamoxifen resistance
(Butt, McNeil et al. 2005, Wierstra and Alves 2006).
3.3.4 Deregulation of FOXM1 negative and positive regulators as potential mechanisms of tamoxifen resistance
The expression pattern of CYCLIN D1 showed a strong increase in its protein
expression in MCF-7 cells overexpressing the active form of FOXM1 compared to
wild-type and tamoxifen resistant cells. It is known that CYCLIN D1/CDK4
phosphorylates pRB releasing FOXM1 from pRB repression (Wierstra and Alves
2006), but CYCLIN D1/CDK4 also phosphorylates FOXM1 in multiple sites (Anders,
Ke et al. 2011). Therefore, it is likely that a positive feedback loop between FOXM1
and CYCLIN D1 occurs in the FOXM1 overexpressing cells. Given the well-
documented role of cyclin D1 in endocrine resistance (Lundgren, Holm et al. 2008,
Wang, Dean et al. 2008, Finn, Dering et al. 2009, Yamashita, Takahashi et al. 2009,
Zwart, Rondaij et al. 2009) and G1/S transition (Fung and Poon 2005, Myatt and Lam
2007, Tashiro, Tsuchiya et al. 2007), our data also support a role for FOXM1 in
mediating breast cancer endocrine sensitivity and resistance at least in part through
modulating CYCLIN D1 expression.
In addition to ERα, ERβ is an oestrogen receptor encoded by a distinct gene
than oestrogen receptor alpha, but both bind with equal affinity oestrogens. ERα
promotes the proliferation of breast epithelium and cancer cells, while ERβ has anti-
proliferative and pro-apoptotic effects. ERβ is expressed in several variants in normal
and malignant tissues, but Roger and colleagues reported a decrease in ERβ protein
expression in pre-invasive mammary tumours compared to normal or benign lesions
(Roger, Sahla et al. 2001). There is a conflicting data regarding the co-expression of
both ERs and association with prognostic, endocrine responsiveness and survival.
However, ERβ has been in general shown to be associated with favourable
prognostic for endocrine therapy in breast cancer. A recent study in our laboratory
has shown FOXM1 has a target of ERβ1, but only in ERα-positive breast cancer
cells. Indeed, I showed by semi-quantitative ChIP that ERβ1 competes with ERα on
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the ERE of FOXM1 promoter to repress FOXM1 transcription (Fig. 3.15).
Consequently, ERβ1 downregulation in tamoxifen resistant cells could lead to
inhibition of cell cycle arrest tamoxifen-induced.
A recent study showed C-MYB as an ERα target gene and determined the role
of C-MYB in the proliferation of ERα positive breast cancer cells, but not in ERα
negative cells (Drabsch, Hugo et al. 2007). While the role of C-MYB has been well
studied in cell growth and transformation, little is known about B-MYB family member.
Although a direct role of B-MYB in cancer has not been yet established, B-MYB is
found amplified in breast, liver, ovarian carcinomas and cutaneous T-cells
lymphomas (Forozan, Mahlamäki et al. 2000, Tanner, Grenman et al. 2000,
Zondervan, Wink et al. 2000, Mao, Orchard et al. 2003). B-MYB expression is
upregulated in metastasis compared to localised prostate tumours and its
overexpression is associated with poor prognosis in breast cancer patients
(Amatschek, Koenig et al. 2004). As FOXM1, B-MYB is a cell cycle-regulatory gene.
Its expression is induced at G0/S and reached a maximum level in S phase (Lam,
Bennett et al. 1995). B-MYB activity is modulated by posttranslational modifications
including phosphorylation during S phase by cyclin/cdk complexes (Sala, Kundu et al.
1997). It has been shown to promote DNA replication and maintenance of genomic
integrity by regulating the transcription of genes essential for G2/M phase
progression (García and Frampton 2006, Tarasov, Tarasova et al. 2008).
Furthermore, B-MYB overexpressing cells were significantly enriched in genes
involved in G2/M progression (Thorner, Hoadley et al. 2009). Based on the
similarities of B-MYB and FOXM1 target genes, a cross-talk between B-MYB and
FOXM1 has been investigated. Recent evidence demonstrated that B-MYB and
FOXM1 co-regulate one another in a positive feedback loop (Lefebvre, Rajbhandari
et al. 2010, Lorvellec, Dumon et al. 2010). ChIP analysis revealed that B-MYB and
FOXM1 co-ordinate the transcriptional regulation of genes involved in proliferation in
germinal centres of B-cell (Lefebvre, Rajbhandari et al. 2010). Therefore, I suggest
that altered B-MYB expression in breast cancer may affect FOXM1 expression and
be involved in tamoxifen resistance. The first western blot analysis on tamoxifen
resistant breast cancer confirmed that B-MYB is deregulated compared to wild-type
MCF-7 cells (Fig. 3.14). In addition, B-MYB has recently been associated with
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tamoxifen resistance in a high-throughput screening for tamoxifen resistance
(Gonzalez-Malerva, Park et al. 2011).
3.3.5 Conclusion
In this study, I showed a differential regulation of FOXM1 in endocrine sensitive
and resistant breast cancer cell lines. These findings also provided potential insights
in the mechanism of anti-oestrogen action and endocrine resistance, and showed
that FOXM1 deregulation may involve deregulated AIB1, CYCLIN D1, ERβ1 or B-
MYB proteins. Furthermore, this study raises a potential new strategy for the
treatment of breast cancer endocrine resistant where FOXM1 is frequently
overexpressed. Targeting FOXM1 with siRNA in this study had a cytostatic effect on
tamoxifen resistant cells. Therefore, the inhibition of FOXM1 with small molecules,
such as thiostrepton or siamycin A, in combination with tamoxifen might resensitise
tamoxifen resistant cells to cell cycle arrest induced by tamoxifen as observed in this
study.
Figure 3.16 Potential pathways in endocrine therapy. Tamoxifen reduces ERα pathway and FOXM1 in endocrine sensitive cells leading to G1 cell cycle arrest. Whereas deregulated proteins including cyclin, proliferative factor, ER co-factor in tamoxifen resistant cells could prevent cell cycle arrest OHT-induced.
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3.4 Future work
This report shows that targeting FOXM1 in tamoxifen resistant breast cancer
cells is an attractive strategy considering that FOXM1 is overexpressed in tamoxifen
resistant breast cancer cells and that FOXM1 deregulation is involved in tamoxifen
resistance. I raised several potential mechanisms of FOXM1 deregulation in this
study that need to be clarified. Treatment with tamoxifen induces the release of ERα
co-activators and the recruitment of ERα co-repressors. However, I observed a high
and constant expression of AIB1 in tamoxifen resistant breast cancer cells.
Consequently, investigating the recruitment, expression and regulation of co-
activators in tamoxifen resistant cells would reveal whether this process is
deregulated and would provide new potential strategies to overcome resistance. For
instance, histone deacetylase inhibitors are already in use as monotherapy or in
combination with taxol and radiation for a wide range of cancers (Dowdy, Jiang et al.
2006, Lane and Chabner 2009, Mueller, Yang et al. 2011). Furthermore, the
electrophile disulfite benzamide DIBA has given promising results in mice models.
DIBA switched tamoxifen agonist to antagonist activity by facilitating the dissociation
of co-activators and the association of co-repressor on the promoter of ER-
responsive genes resulting in a decrease in xenograft tumor growth of tamoxifen
resistant human cells in mice (Wang, Yang et al. 2006). This study also reveals a
potential positive feedback loop between FOXM1 and CYCLIN D1. It was reported
that CYCLIN D1 modulates the phosphorylation status of pRB leading to the release
of FOXM1 by pRB (Gladden and Diehl 2003, Wierstra and Alves 2006). CYCLIN D1
is a validated anti-cancer target with several compounds in clinical trials. Therefore,
further investigations of FOXM1 regulation by CYCLIN D1 and reciprocal would give
new opportunities for treating tamoxifen resistant breast cancer patients. Finally, the
study of ERβ1 in our laboratory identified ERβ1 as a negative FOXM1 regulator that
could be downregulated in tamoxifen resistant cells. ERβ1 was observed decreased
in pre-invasive mammary tumour compared to normal or benign lesions, but so far
altered expression of ERβ1 in tamoxifen resistance has not been investigated
(Roger, Sahla et al. 2001). Thereby, the study of ERβ1 expression would provide
further molecular mechanism of tamoxifen resistance and potential strategy to
overcome tamoxifen resistance. B-MYB has been recently identified as a positive
regulator of FOXM1, which co-ordinates with FOXM1 to regulate genes involved in
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G2/M phase transition. Our preliminary data suggest that B-MYB is also
overexpressed in tamoxifen resistant ERα positive breast cancer cells and might
participate to FOXM1 deregulation via a feedback loop. Therefore, it would be
interesting to investigate B-myb role and regulation in tamoxifen resistance.
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CHAPTER 4 ATM and p53 regulate FOXM1 expression via E2F in
breast cancer epirubicin treatment and resistance
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4.1 Introduction
Cytotoxic chemotherapy agents are the most commonly drugs used in cancers.
Taxanes and anthracyclines are cytotoxic chemotherapy drugs that have been
frequently used in the neo-adjuvant and adjuvant settings to reduce tumour size prior
to surgery and eliminate left over cells to prevent recurrence (Martin, Villar et al.
2003). These cytotoxic agents are used to treat patients with ER/PR/HER2 receptors
in combination to anti-oestrogen or HER2-targeted therapies. Importantly,
chemotherapy drugs are the only therapeutic option for triple receptor negative
patients. Furthermore, cytotoxic chemotherapy agents are used to treat breast cancer
patients that are resistant to endocrine and targeted therapies, and these agents are
particularly important in the treatment of advanced or metastatic solid cancers
(Alvarez 2010, Palmieri, Krell et al. 2010).
Anthracyclines, including doxorubicin (also called Daunorubicin) and epirubicin,
are a group of Streptomyces peucetius bacteria-derived antibiotics commonly used in
cancer chemotherapy. These compounds have been shown to be effective for the
treatment of a broad spectrum of cancers such as breast, lung, and ovary
carcinomas as well as leukaemia (Lown 1993, Nielsen, Maare et al. 1996). Despite
being some of the most effective and widely used anti-cancer drugs in the clinic,
patients relapse because of the development of acquired drug resistance (Gonzalez-
Angulo, Morales-Vasquez et al. 2007, Broxterman, Gotink et al. 2009, Zelnak 2010).
The exact mechanism of action of anthracyclines is still not completely clear, but
likely to interfere with DNA replication and induce DNA intercalation triggering DNA
damages (Gewirtz 1999, Rivera 2010). Resistance to these DNA targeting anti-
cancer drugs is a major clinical obstacle for patients that initially respond to the
treatment. It involves multiple mechanisms including enhancement of DNA repair.
Consistently, DNA repair gene network signature has been found to be associated
with anthracycline response in triple negative metastatic breast cancer (Rodriguez,
Makris et al. 2010). A better understanding of the molecular mechanisms of
anthracycline action and resistance is required for the development of novel
strategies for the treatment of advanced or metastatic breast cancer and for
overcoming resistance.
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FOXM1 is required for normal G1/S and G2/M cell cycle phase transitions.
Besides its involvement in cell cycle transitions, FOXM1 has a multifaceted role in
biological processes. Notably, FOXM1 has been recently linked to DNA damage
repair (Tan, Raychaudhuri et al. 2007, Kwok, Peck et al. 2010). In addition, FOXM1
dysregulation has been shown to be involved in the development of cisplatin
resistance in breast cancer (Kwok, Peck et al. 2010). Accordingly, FOXM1
overexpression has been shown to confer resistance to the humanized anti-HER2
monoclonal antibody (trastuzumab) and microtubule-stabilizing drug (paclitaxel)
(Carr, Park et al. 2010). Moreover, chapter 3 of this thesis shows that FOXM1 is a
transcriptional target of ER and play key role in breast cancer endocrine therapy
resistance (Millour, Constantinidou et al. 2010). In this chapter, I investigated the
expression and regulation of FOXM1 in epirubicin sensitive and resistant MCF-7
breast carcinoma cell lines and its involvement in epirubicin resistance.
4.2 Transcriptional regulation of FOXM1 by p53 in epirubicin sensitive MCF-7 cells
4.2.1 Activation of p53 transcriptionally represses FOXM1
The recent observation that p53 represses FOXM1 expression following
daunorubicin treatment led to predict that epirubicin also activates p53 to repress
FOXM1 expression in breast cancer cells (Barsotti and Prives 2009). To assess the
role and mechanism by which p53 mediates the epirubicin response in breast cancer
cells, I first examined the expression of FOXM1 in p53 positive MCF-7 and p53
negative MDA-MB-453 breast cancer cell lines. Western blot analysis of MCF-7 cells
revealed that epirubicin treatment strongly induced the expression of the p53 protein
and its target the cyclin-dependent kinase inhibitor p21Cip1 from 16 h post-treatment,
and decreased FOXM1 protein level significantly from 24 h post-treatment (Fig. 4.1).
Unsurprisingly, p53 and the inhibitor p21Cip1 were undetectable in MDA-MB-453 cell
line before and after treatment. Consistently, RT-qPCR analysis revealed no
significant decrease in FOXM1 transcript level in MDA-MB-453 cells, while epirubicin
induced a drastic reduction of FOXM1 mRNA level in MCF-7 cells. Contrary to
FOXM1 mRNA level, FOXM1 protein levels varied throughout the treatment.
However, the variation in FOXM1protein levels can be attributed to multiple levels of
regulation and a shorter division time in these cell rather than treatment related.
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Indeed, FOXM1 protein level varies throughout the cell cycle as well as cyclin A and
B1 (Fig. 4.1) and is subject to post-translational modifications including
phosphorylations. Collectively, these results show that FOXM1 is downregulated at
mRNA and protein levels in response to epirubicin in the p53 positive MCF-7 cells,
while FOXM1 expression remained relatively constant in the p53 negative MDA-MB-
453 cells, suggesting that activated p53 plays a role in FOXM1 regulation.
To further confirm that p53 is responsible for the downregulation of FOXM1
expression in MCF-7 cells following epirubicin treatment, MCF-7 cells were
transiently transfected with non-specific (NS siRNA) or p53-targeting siRNA (p53
siRNA), treated with epirubicin and FOXM1 expression examined. Western blot and
RT-qPCR analysis showed that silencing of p53 attenuated FOXM1 downregulation
at both protein and mRNA levels in response to epirubicin (Fig. 4.2A and 4.2C). The
inability of p53 depletion to completely abolish the downregulation of FOXM1 also
suggested that p53 might not be the sole regulator of FOXM1 expression in response
to epirubicin (Fig. 4.2A). A previous study showed that p53 represses FOXM1
expression via pRB following daunorubicin treatment (Barsotti and Prives 2009).
Thereby, one mechanism by which p53 can repress FOXM1 expression is through its
ability to induce p21Cip1, which can in turn repress cyclin-CDK-mediated pRB
hyperphosphorylation, resulting in the repression of E2F transcriptional activity
(Giacinti and Giordano 2006). Surprisingly, although p53 knock-down abrogated the
induction of p21Cip1 and the downregulation of FOXM1 by epirubicin, silencing of
p21Cip1 had little effect on the epirubicin-induced FOXM1 downregulation, suggesting
that epirubicin can also repress FOXM1 expression via p21Cip1-independent
mechanisms (Fig. 4.2B and 4.2D). To further investigate the role of p53 and p21Cip1
in regulating FOXM1 expression in response to epirubicin, wild-type (wt), p53-
deficient (p53-/-), and p21-deficient (p21Cip1-/-) mouse embryo fibroblasts (MEFs) were
subjected to epirubicin treatment and the expression of FOXM1 investigated (Fig.
4.3). Treatment of the wt and p21Cip1-/- MEFs with epirubicin resulted in a reduction of
FOXM1 mRNA expression within 16 h, further confirming that p21Cip1 is not essential
for the repression of FOXM1 expression by epirubucin (Fig. 4.3). In contrast,
epirubicin did not cause a downregulation of FOXM1 mRNA expression in the p53-
deficient MEFs (Fig. 4.3). Together these data support the idea that epirubicin
represses FOXM1 expression at the transcriptional level through p53.
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Figure 4.1 Expression of FOXM1 in response to epirubicin treatment in breast cancer cell lines. MCF-7 and MDA-MB-453 cells cultured in 10 % FCS and phenol red DMEM medium were treated with 1 µmol/L of epirubicin. At times indicated, cells were collected and analysed for FOXM1, CYCLIN A, p53, p21Cip1, CYCLIN B1 and β-tubulin expression by western blotting. FOXM1 mRNA levels of these cells were also analysed by RT-qPCR, and normalized with L19 RNA expression. All data shown represent the averages of data from three independent experiments, and the error bars show the standard deviations. Statistical analyses were done using Student’s t test. *, P≤0.1, **, P≤0.01 and ***, P≤0.001, significant and n.s, non significant.
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Figure 4.2 Activation of p53 in MCF-7 cells represses FOXM1 expression. MCF-7 cells cultured in 10 % FCS and phenol red DMEM medium were either transfected with non-specific siRNA (NS siRNA), siRNA smart pool against p53 (p53 siRNA) (A. and C.), or siRNA smart pool against p21Cip1 (p21Cip1 siRNA) (B. and D.) (100 nmol/L). Twenty-four hours after transfection, MCF-7 cells were treated with 1 µmol/L of epirubicin and harvested for western blot and analysed using RT-qPCR at 0, 24 and 48 h. The protein expression levels were determined for FOXM1, p53, p21Cip1 and β-tubulin and the mRNA level was determined for FOXM1 and normalized with L19 RNA expression. Columns, means derived from three independent experiments; bars, SD. Statistical analyses were done using Student’s t test. *, P≤0.1, **, P≤0.01 and ***, P≤0.001, significant and n.s, non significant.
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Figure 4.3 FOXM1 repression by p53 in a p21-independent manner. Wild-type, p53-/- and p21Cip1-/- MEF cells cultured in 10 % FCS and phenol red DMEM medium were treated with 1 µmol/L of epirubicin for 0, 16, 24 and 48 h, and RT-qPCR was performed to determine FOXM1 mRNA transcript levels and normalize with L19 RNA expression. Columns, means derived from three independent experiments; bars, SD. Statistical analyses were done using Student’s t test. *, P≤0.1 and ** P≤0.01, significant.
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4.2.2 p53 can regulate FOXM1 through an E2F site in its promoter
The pRB/E2F transcription factors are principal regulators of the cell cycle and
function downstream of the p53 canonical pathway (Giacinti and Giordano 2006). To
assess whether the E2F transcription factors are involved in the p53-dependent
FOXM1 repression, we analysed the expression pattern of E2F1, a well-
characterized E2F-responsive gene product as well as a subunit of the E2F
transcription factor dimers (DeGregori and Johnson 2006). The other E2F family
members are expressed in different tissues and contexts (Kusek, Greene et al.
2000). Treatment of MCF-7 cells with epirubicin markedly reduced E2F1 mRNA
levels within 16 h, whereas E2F1 transcript level increased in response to epirubicin
in the p53 negative MDA-MB-453 cells (Fig. 4.4A). Furthermore, the close correlation
between the mRNA expression pattern of E2F1 and FOXM1 in MCF-7 cells suggests
that p53 is likely to downregulate FOXM1 expression through the repression of E2F
activity.
To provide further evidence that epirubicin represses FOXM1 expression
through inhibition of E2F activity, MCF-7 cells were treated with epirubicin for 0 and
24 h, followed by ChIP analyses of E2F1 and its negative regulator pRB on FOXM1
promoter (Fig. 4.4B). Our semi-quantitative ChIP assay showed that the in vivo
occupancy of the proximal FOXM1 promoter by E2F1 decreased and pRB increased
after epirubicin treatment, indicating that epirubicin causes the depletion of the
transactivator E2F and the accumulation of the transcriptionally repressive pRB
protein on FOXM1 promoter (Fig. 4.4B). Although, this experiment indicated a
decreased in E1F and an increased in pRB occupancies on FOXM1 promoter, a
ChIP assay using a quantitative PCR method would provide the exact quantification
of the recruitment of these proteins.
We next analysed the involvement of the putative E2F-binding sites in FOXM1
promoter in FOXM1 repression upon epirubicin treatment. To this end, MCF-7 cells
were transiently transfected with a luciferase reporter (pGL3) driven by either a 2.4
kbp (Full Length), a 1.4 kbp (HindIII), or a 296 bp (ApaI) FOXM1 promoter, and the
promoter activity was assayed at 0, 24 and 48 h after epirubicin treatment (Fig. 4.5).
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The activity of all three FOXM1 promoter constructs was markedly reduced following
exposure to 1 µmol/L epirubicin, consistent with the fact that the putative E2F-binding
sites (site 1: -58 bp and site 2: -24 bp) locate inside all three FOXM1 promoter
constructs (Fig. 4.5). We next examined whether p53 exerts its repression on FOXM1
promoter activity through these putative E2F-binding sites. To this end, we co-
transfected into MCF-7 cells increasing amounts of pcDNA3-Flag-p53 together with
either the wild-type ApaI FOXM1 promoter reporter (WT) or the ApaI FOXM1
promoter lacking one (E2Fmut1 or E2Fmut2) or both (E2Fmut1/2) putative E2F-
binding sites. The results showed that p53 caused a drastic reduction (12.7 fold) in
E2Fmut1 luciferase activity, comparable to that observed for WT (11.5 fold) (Fig.
4.6A). By contrast, the repression by p53 was considerably reduced in both the
E2Fmut2 and the E2Fmut1/2, suggesting that the second putative E2F-binding site
(site 2) mediates the repression of FOXM1 promoter by p53. Next, activity of the wild-
type ApaI (WT) as well as mutated pGL3-ApaI constructs (E2Fmut1, E2Fmut2, and
E2Fmut1/2) was examined by co-transfection assays in MCF-7 cells with different
amounts of pCMV-E2F1 expression vector. The results showed that the E2Fmut1
construct showed similar responsiveness to E2F1 as the WT (Fig. 4.6B). In contrast,
both the E2Fmut2 and the E2Fmut1/2 mutants lost the majority of their
responsiveness to E2F1 transfection. Together these co-transfection results provide
strong evidence that the E2F-binding element located at −24 bp confers the
responsiveness to p53 and E2F1. Taken together, these results indicate that
epirubicin can induce p53 to repress FOXM1 through modulating E2F activity on
FOXM1 promoter.
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Figure 4.4 E2F1 is decreased in response to epirubicin in MCF-7 cells. A. MCF-7 and MDA-MB-453 cells cultured in 10 % FCS and phenol red DMEM medium were treated with 1 µmol/L of epirubicin for 0, 16, 24 and 48 h and RT-qPCR was performed to determine E2F1 transcript levels and normalize with L19 RNA expression. Columns, means derived from three independent experiments; bars, SD. Statistical analyses were done using Student’s t test. *, P≤0.1 and ** P≤0.01, significant. B. MCF-7 cells untreated or treated with 1 µmol/L of epirubicin for 24 h were used for ChIP assays using IgG negative control, anti-E2F1 and anti-pRB antibodies as indicated. After crosslink reversal, the co-immunoprecipitated DNA was amplified by PCR using primers amplifying the FOXM1 E2F-binding sites containing region (-184/+4 bp) and a control region (-1157/-1257 bp), and resolved in 1 % agarose gel.
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Figure 4.5 FOXM1 promoter activity in response to epirubicin. Schematic representation of the full length, HindIII and ApaI FOXM1-luciferase reporter constructs and the E2F-binding sites 1 (-58 bp) and 2 (-24 bp). MCF-7 cells were transiently transfected with 20 ng of either the empty pGL3-basic, pGL3-Full length, pGL3-HindIII or the pGL3-ApaI, and cells were treated with 1 µmol/L of epirubicin. Cells were, as described in material and methods, harvested at 0, 24 and 48 h after treatment and assayed for luciferase activity. All relative luciferase activity values are corrected for co-transfected Renilla activity. The fold of repression were calculated between 0 h and 48 h of epirubicin treatment. Columns, means derived from three independent experiments; bars, SD.
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Figure 4.6 Modulation of FOXM1 promoter by p53 and E2F1 via E2F binding site. MCF-7 cells cultured in 10 % FCS and phenol red DMEM medium were transiently transfected with 20 ng of either the wild-type (WT), E2F-binding site 1 mutated (E2Fmut1), E2F-binding site 2 mutated (E2Fmut2), or E2F-binding site 1 and 2 mutated (E2Fmut1/2) pGL3-ApaI constructs together with increasing amounts (0, 10 and 30 ng) of pcDNA3-Flag-p53 (A.) and pCMV-E2F1 (B.). Cells were harvested after 24 h transfection and assayed for luciferase activity as described in Material and Methods. All relative luciferase activity values are corrected for co-transfected Renilla activity. The fold of repression and activation were calculated and indicated between 0 h and 48 h of epirubicin treatment. Columns, means derived from three independent experiments; bars, SD. Statistical analyses were done using Student’s t test. **, P≤0.01 and ***, P≤0.001, significant and n.s, non significant.
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4.3 Differential mechanism of FOXM1 regulation in epirubicin resistant MCF-7 cells
4.3.1 Deregulation of FOXM1 protein and mRNA levels in epirubicin resistant cells
The involvement of FOXM1 in DNA damage response and chemotherapy drug
resistance led us to hypothesise that FOXM1 has a role in anthracycline sensitivity as
well as resistance in breast cancer. In order to test this conjecture, an epirubicin
resistant breast cell line MCF-7EPIR was used for this study. MCF-7EPIR cell line was
established by a former PhD student by chronic exposure of the parental drug
sensitive MCF-7 to stepwise increases in epirubicin concentration until a
concentration of resistance up to 10 µmol/L. I confirmed by SRB assays that MCF-
7EPIR cells displayed strong resistance to cell death epirubicin-induced compared to
the parental MCF-7 cells (Fig. 4.7A). I next examined the effect of epirubicin on the
cell viability of MCF-7 and MCF-7EPIR cells at 1 µmol/L, a concentration generally
used in cancer therapy. The SRB assay revealed that survival of MCF-7 cells was
significantly inhibited following epirubicin treatment, while survival of MCF-7EPIR cells
was relatively unaffected in the presence of epirubicin (Fig. 4.7B). There was also a
significant difference in the survival rate between the epirubicin-treated MCF-7 and
MCF-7EPIR cells at both 24 h and 48 h. Cell cycle analysis showed that epirubicin
exposure (1 µmol/L) induced an accumulation of MCF-7 cells at G2/M and sub-G1
phases, indicative of G2/M phase transition delay and cell death, whereas no
significant changes in cell cycle profile are observed for MCF-7EPIR cells (Fig. 4.7C).
Subsequent western blot analysis revealed no significant changes in the levels
of FOXM1 and FOXM1 protein targets, CYCLIN B1 and PLK, following 48 h
treatment with epirubicin (1 µmol/L) in MCF-7EPIR cells in contrast to the
downregulation observed in MCF-7 cells Figure 4.1 (Fig. 4.8A). Consistently, RT-
qPCR analysis revealed no significant decrease in FOXM1 transcript level in MCF-
7EPIR cells (Fig. 4.8B). Importantly, p53 and p21Cip1 protein levels were undetectable
in MCF-7EPIR cells by western blot analysis (Fig. 4.8A), the band detected in the p53
lane being an unspecific band. Although p53 mRNA levels are not relevant for its
functional activity, we further investigated whether MCF-7EPIR cells could have lost
p53 mRNA expression. RT-qPCR analysis further showed that p53 transcript level is
reduced by 3.5 fold in MCF-7EPIR cells compared to MCF-7 cells (Fig. 4.8C).
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Collectively, these results show that FOXM1 expression is deregulated in epirubicin
resistant MCF-7 cells likely due to the lost of repression by p53, suggesting that
FOXM1 has a role in epirubicin sensitivity and resistance.
4.3.2 Increased DNA repair in epirubicin resistant cells
Next, I sought to determine the molecular mechanism that confers epirubicin
resistance to MCF-7EPIR cells. It has been previously shown that FOXM1 expression
is associated with cisplatin-induced DNA damage response and drug resistance
(Kwok, Myatt et al. 2008). I therefore examined the formation of DNA damage foci by
P-H2AX staining in MCF-7 and MCF-7EPIR cells following epirubicin treatment,
including some enlargement of cells showed by the white arrows and represented in
the white window. The results showed an increase in the mean number of P-H2AX
foci/cell over time after epirubicin treatment in MCF-7 cells, while the level of P-H2AX
foci/cell remained relatively constant in MCF-7EPIR cells, suggesting higher DNA
repair activities in these cells (Fig. 4.9). This result was also confirmed in a recent
study (Monteiro, Khongkow et al. 2012). To investigate this further, we evaluated the
expression level of the DNA repair protein ATM in MCF-7 and MCF-7EPIR cells.
Western blot and RT-qPCR analysis demonstrated that the levels of ATM protein and
mRNA are strongly upregulated in MCF-7EPIR cells compared to MCF-7 cells (Fig.
4.10A), thus suggesting a role of ATM in mediating an increase in DNA repair activity
in resistant cells. The ATR mRNA expression level was investigated and showed an
significantly elevated ATR mRNA level in MCF-7 cells compared with MCF-7EPIR
cells. This finding might suggest a difference in the activation of DNA repair proteins
between these two cell lines (Fig. 4.10B).
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Figure 4.7 Characterisation of epirubicin resistant MCF-7EPIR cells. A. MCF-7 and MCF-7EPIR cells cultured in 10 % FCS and phenol red DMEM medium were treated with increasing concentrations of epirubicin for 24 h. Number of cells was measured using SRB assay as described in Material and Methods. B. MCF-7 and MCF-7EPIR cells cultured in 10 % FCS and phenol red DMEM medium were treated with 1 µmol/L of epirubicin for 0, 16, 24 and 48 h and SRB assay was performed. Statistical analyses were realized using Student t-test for untreated versus treated. C. MCF-7 and MCF-7EPIR cells cultured in 10 % FCS and phenol red DMEM medium were treated with 1 µmol/L of epirubicin for 0, 16, 24 and 48 h and cells were stained with propidium iodide and analysed FACS analysis carried out. Percentage of cells in each phase (sub-G1, G1, S, G2/M) is indicated. Representative data from three independent experiments are shown.
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Figure 4.8 Inverse correlation between FOXM1 and p53 expression in MCF-7 and MCF-7EPIR cell lines. A. and B. MCF-7EPIR cells cultured in 10 % FCS and phenol red DMEM medium were treated with 1 µmol/L of epirubicin for 0, 16, 24 and 48 h. At indicated time, cells were collected and analysed by western blotting to determine the protein expression levels of FOXM1, CYCLIN B1, PLK, p53, p21Cip1 and β-tubulin (A.), and by RT-qPCR (B.) to determine FOXM1 mRNA transcript levels. Columns, means derived from three independent experiments; bars, SD. C. MCF-7 and MCF-7EPIR cells cultured in 10 % FCS and phenol red DMEM medium were harvested to determine p53 mRNA transcript level by RT-qPCR. Columns, means derived from three independent experiments; bars, SD. Statistical analyses were done using Student’s t test. **, P≤0.01 and ***, P≤0.001, significant and n.s, non significant.
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Figure 4.9 Epirubicin resistant MCF-7EPIR cells show a reduction of DNA damage in response to epirubicin treatment. A. MCF-7 and MCF-7EPIR cells treated with 1 µmol/L of epirubicin for 0, 0.5, 1.5 and 5 h were stained with P-H2AX antibody and DAPI. Images were visualized and scored by ImageXpress (Molecular Devices). The results are the average of three independent experiments. Mean ± SD. Statistical analyses were performed using Students’s test. **, P ≤ 0.01 significant; n.s non significant. B. MCF-7 and MCF-7EPIR cells treated with 1 µmol/L of epirubicin were stained with P-H2AX antibody (green) and DAPI (red). Images visualized by confocal microscopy. Images: magnification: x 20; insets x 80.
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Figure 4.10 Increased expression of ATM in epirubicin resistant MCF-7EPIR cells. MCF-7 and MCF-7EPIR cells cultured in 10 % FCS and phenol red DMEM medium were analysed for FOXM1, ATM and and β-tubulin by western blotting (A.). Cells were harvested to determine ATM (B.) and ATR (C.) mRNA levels using RT-qPCR. Columns, means derived from three independent experiments; bars, SD. Statistical analyses were done using Student’s t test. **, P≤0.01 and ***, P≤0.001, significant and n.s, non significant.
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4.3.3 ATM is involved in FOXM1 regulation and epirubicin resistance
To determine whether the ATM signalling pathway is involved in FOXM1
regulation in response to epirubicin, we treated MCF-7 and MCF-7EPIR cells with
epirubicin in the absence or presence of Ku-55933, a known ATM inhibitor (Fig.
4.11). Western blot analysis demonstrated that epirubicin induced a shift of FOXM1
protein at a lower size in MCF-7 cells as already shown previously, while it did not
affect FOXM1 protein level and/or size in MCF-7EPIR cells (Fig. 4.11). However, the
combination of epirubicin with Ku-55933 repressed E2F1 and FOXM1 protein
expression in MCF-7 and MCF-7EPIR cells, indicating that Ku-55933 re-sensitised
the resistant MCF-7EPIR cells to FOXM1 downregulation epirubicin-induced (Fig.
4.11). These results suggest that the ATM DNA damage response is involved in
FOXM1 regulation in MCF-7EPIR cells independent of p53 status. However, it has
been shown that Ku-55933 alone has no effect on cell cycle while Ku-55933
combined with etoposide induces a G2 arrest (Hickson et al. 2004). Taking into
account that FOXM1 expression is reduced when cell cycle is stopped, FOXM1
downregulation Figure 4.11 might be the consequence of Ku-55933 combined with
epirubicin treatment.
Consequently, ATM expression and activity were investigated in MCF-7 and
MCF-7EPIR cells by western blot analysis (Fig. 4.12A). Treatment with epirubicin
activated ATM phosphorylation (on serine 1981) and also induced ATM expression in
MCF-7EPIR within 24 h, while this induction was not detectable in MCF-7 cells (Fig.
4.12A). Phosphorylation of ATM downstream target CHK2 was strongly enhanced in
MCF-7EPIR cells and to a much lesser extent in MCF-7 cells. In addition, treatment
with epirubicin strongly activated p53 reflected by the phosphorylation of p53 on
serine 15. In contrast, phosphorylated p53 on serine 15 was undetectable in MCF-
7EPIR cells, demonstrating that p53 is not active in these cells.
To determine whether ATM is a key player of FOXM1 regulation, I silenced
ATM expression using siRNA strategy (ATM siRNA) in both MCF-7 and MCF-7EPIR
cells, and studied FOXM1 expression in response to epirubicin (Fig. 4.12B). Western
blot analysis showed that ATM knock-down had little effect on FOXM1 and E2F1
expression in MCF-7 cells. While the expression level of FOXM1 remained constant
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in MCF-7EPIR cells transfected with non-specific siRNA (NS siRNA) upon epirubicin
treatment, epirubicin caused a decrease in FOXM1 protein expression in MCF-7EPIR
cells when ATM is silenced (Fig. 4.12B). Furthermore, E2F1 protein decreased in
MCF-7EPIR cells following ATM knock-down and epirubicin treatment, which
correlates with FOXM1 protein expression. These findings confirmed a differential
mechanism of regulation by ATM between MCF-7 and MCF-7EPIR cells. These
findings also suggest that the lack of active p53 and the induction of ATM in MCF-
7EPIR cells are responsible for FOXM1 expression in response to epirubicin.
As ATM is a protein kinase that phosphorylates proteins including p53, mdm2,
h2ax, chk2 following DNA damage, I performed a time course with MCF-7 and MCF-
7EPIR cells treated with epirubicin (Zhou and Elledge 2000). I showed that FOXM1
protein level (higher band) increased in MCF-7EPIR cells, while FOXM1 is
downregulated in MCF-7 cells (Fig. 4.13A). Furthermore, I immunoprecipitated
FOXM1 proteins and probed with anti-MPM2, an antibody recognising
phosphorylated proteins, to investigate FOXM1 phosphorylation status in MCF-7 and
MCF-7EPIR cells epirubicin-treated. Immunoblotting with MPM2 antibody showed a
decrease in phosphorylation of immunoprecipitated FOXM1 in MCF-7 cells, while
FOXM1 phosphorylation remained high in MCF-7EPIR cells following epirubicin (Fig.
4.13B). In addition, it has previously been shown that FOXM1 protein is
phosphorylated by CHK2 after DNA damage. Given that CHK2 functions directly
downstream of ATM in DNA damage response, it is predicted that the induction of
FOXM1 expression by ATM may occur through post-translational mechanisms (Tan,
Raychaudhuri et al. 2007). In contrast to Tan et al. study, silencing of both checkpoint
kinases (CHK1 and CHK2) did not affect FOXM1 protein expression in untreated and
treated MCF-7EPIR cells (Fig. 4.13C). Notably, the loading of CHK1 siRNA protein
samples was lower than non-specific and CHK2 siRNA, but FOXM1 levels still
remained steady in these samples over the time course.
To further investigate FOXM1 regulation by ATM, I used the siRNA strategy to
reduce ATM expression (ATM siRNA) and investigated FOXM1 mRNA transcript
levels in MCF-7EPIR cells treated with epirubicin. ATM silencing significantly reduced
ATM mRNA levels as well as FOXM1 mRNA levels in MCF-7EPIR cells treated with
epirubicin, suggesting that ATM regulates FOXM1 at transcriptional level (Fig.
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4.14A). Given that E2F1 protein expression followed FOXM1 protein expression
pattern when ATM is silenced, I analysed E2F1 mRNA levels in MCF-7EPIR cells
treated with epirubicin and performed ChIP assay. The results showed that E2F1
mRNA expression and E2F1 occupancy of FOXM1 promoter remained steady in
MCF-7EPIR cells treated with epirubicin (Fig. 4.14B and 4.14C).
These data suggested that ATM regulates FOXM1 transcriptionally via E2F1
and that FOXM1 might also be regulated by phosphorylations in MCF-7EPIR cells.
Given the role of ATM in DNA repair and the fact that ATM regulates FOXM1 in
epirubicin resistant cells, it is likely that FOXM1 has a role in epirubicin resistance. In
addition, the role of FOXM1 in epirubicin sensitivity and resistance is further
supported by the observations that overexpression of FOXM1 in MCF-7 cells can
decrease the sensitivity to epirubicin (supplementary data realised by Julia K. Langer
Fig. S.D.7.3) and that FOXM1 knock-down in MCF-7EPIR cells did mimic the
cytotoxic effects of epirubicin on MCF-7 cells (Fig. 4.15). However, the preliminary
SRB result did not show that ATM silencing has an effect on the survival of the
epirubicin resistant cells compared to FOXM1 silencing (Fig. 4.15). These results
could indicate that FOXM1 is regulated by multiple pathways including ATM, but
remains the main regulator in epirubicin resistance.
Although the SRB assay showed that FOXM1 silencing reduced the survival of
the epirubicin resistance cells, a clonogenic or colony formation assay would be a
better method to test the effect of FOXM1 inhibition on epirubicin sensitivity and
resistance. Clonogenic assay is the method of choice to determine cell reproductive
death after treatment. Only a fraction of seeded cells retains the capacity to produce
colonies (Franken, Rodermond et al. 2006). Testing FOXM1 silencing in epirubicin
resistant cells using this assay would tell us whether FOXM1 is a key player for
epirubicin resistance.
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Figure 4.11 ATM inhibition and epirubicin downregulate FOXM1 in MCF-7EPIR cells. MCF-7 and MCF-7EPIR cells were treated with 1 µmol/L of epirubicin alone or in combination with 10 µmol/L of Ku-55933 for 24 h and the protein expression levels of FOXM1, P-CHK2, CHK2, E2F1 and β-tubulin were analysed by western blot analysis.
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Figure 4.12 ATM is involved in FOXM1 regulation in epirubicin resistant MCF-7EPIR cells. A. MCF-7 and MCF-7EPIR were treated with 1 µmol/L of epirubicin and the protein expression levels of P-ATM, ATM, P-CHK2, CHK2, P-p53 (ser15) and β-tubulin were analysed by western blot analysis. B. MCF-7 and MCF-7EPIR cells were either transfected with non-specific (NS) siRNA (100 nmol/L) or siRNA smart pool against ATM (100 nmol/L). Twenty-four hours after transfection, cells were treated with 1 µmol/L of epirubicin and harvested for western blot at 0, 24 and 48 h. The protein expression levels were determined for FOXM1, ATM, E2F1, cleaved PARP and β-tubulin.
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Figure 4.13 Phosphorylation of FOXM1 in MCF-7EPIR cells. A. MCF-7 and MCF-7EPIR cells cultured in 10 % FCS and phenol red medium were treated with 1 µmol/L of epirubicin in a time course of 48 h. Cell lysates were prepared at indicated times, and the expression of FOXM1 and β-tubulin was analysed by western blotting. B. MCF-7 and MCF-7EPIR cells cultured in 10 % FCS and phenol red medium were treated with 1 µmol/L of epirubicin for 24 h. Cell lysates were subjected to immunoprecipitation using anti-FOXM1 antibody and immunoblotted with anti-MPM2 and FOXM1 antibodies. C. MCF-7EPIR cells were either transfected with non-specific (NS) siRNA, CHK1- or CHK2-targeting siRNA (100 nmol/L) and treated with 1 μmol/L of epirubicin for 0, 24 and 48 h. The protein levels were analysed by western blotting using anti-FOXM1, anti-PLK, anti-CHK1, anti-CHK2 and anti-β-tubulin antibodies.
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Figure 4.14 E2F1 occupancy on FOXM1 promoter remains steady in MCF-7EPIR cells. A. Cells were transfected with non-specific and FOXM1-targeting siRNA (100 nmol/L) and were treated with 1 μmol/L of epirubicin for 48 h and harvested for determination of FOXM1 and ATM mRNA levels by RT-qPCR analysis. Statistical analyses were performed using Students’s test. **, P ≤ 0.01 significant; n.s non significant. B. MCF-7EPIR cells were treated with 1 µmol/L of epirubicin for 0, 16, 24 and 48 h and RT-qPCR was performed to determine E2F1 transcript levels. Columns, means derived from three independent experiments; bars, SD. C. After cross-link reversal, the co-immunoprecipitated DNA was amplified by PCR using primers amplifying the FOXM1 E2F1-biding sites containing region (-184/+4) and a control region (-1157/-1257), and resolved on 2 % agarose gel (left panel). Quantification by RT-qPCR gave similar results (right panel).
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Figure 4.15 Silencing of FOXM1 combined with epirubicin treatment increases cell death in MCF-7EPIR cells. A. MCF-7EPIR cells were either transfected with non-specific (NS) siRNA or FOXM1-targeting siRNA (100nmol/L) and treated with 1μmol/L of epirubicin for 72 h. Cells were harvested for western blot analysis to validate the silencing efficiency using anti-FOXM1 and anti-β-tubulin antibodies. B. The transfected MCF-7EPIR cells treated with 1μmol/L of epirubicin were collected for SRB assay at 0, 24, 48, 72 h. C. The transfected MCF-7EPIR cells treated with 1μmol/L of epirubicin were also collected for FACS analysis carried out after propidium iodide staining. Cell death was analysed using flow cytometry. The percentages of cells in sub-G1 were calculated. Columns, means derived from three independent experiments; bars, SD. D. MCF-7EPIR cells were either transfected with non-specific (NS) siRNA or ATM-targeting siRNA (100nmol/L) and treated with 1μmol/L of epirubicin for 72 h. The transfected MCF-7EPIR cells treated with 1 μmol/L of epirubicin were collected for SRB assay at 0, 24, 48 h. Experiment performed once in triplicates.
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4.4 Discussion
4.4.1 FOXM1 is a crucial target of p53
FOXM1 has been found to be frequently upregulated in a large variety of human
cancers (Wang, Kiyokawa et al. 2002, Kalinichenko, Major et al. 2004, Kalin, Wang et
al. 2006, Kim, Ackerson et al. 2006, Liu, Dai et al. 2006, Laoukili, Stahl et al. 2007,
Wang, Banerjee et al. 2007). In addition, emerging evidence revealed that FOXM1
also has a role in cancer drug resistance. Studies demonstrated that FOXM1 level is
an important determinant of sensitivity to breast cancer chemotherapy drugs, such as
trastuzumab, gefitinib, lapatinib, paclitaxel and cisplatin (Kwok, Peck et al. 2010).
Consistent with these findings, this study established that FOXM1 is a crucial cellular
target of the anthracycline epirubicin in breast cancer cells. FOXM1 expression is
downregulated by epirubicin in the sensitive MCF-7 cells, but not in the resistant
MCF-7EPIR cells. Moreover, FOXM1 protein levels are higher in the epirubicin
resistant MCF-7EPIR cells relative to the sensitive MCF-7 cells. Taken together,
these data suggest that FOXM1 also has a role in epirubicin resistance. In
agreement, a recent study revealed that the anthracycline daunorubicin can repress
FOXM1 expression through the sequential activation of p53, p21Cip1 and RB family of
proteins (Barsotti and Prives 2009). Using p53-/- and wt MEFs, we established that
FOXM1 expression is negatively regulated by p53 (Fig. 4.3). However, epirubicin can
effectively repress FOXM1 expression in the p21Cip1-/- MEFs (Fig. 4.3). This finding
indicates that p53 can repress E2F activity and FOXM1 expression independent of
the cyclin-dependent kinase inhibitor p21Cip1, despite previous studies showing that
the activation of pRB by the anthracycline daunorubicin is mediated at least partially
through p21Cip1 (Barsotti and Prives 2009). Based on the fact that E2F1 gene is an
E2F-regulated gene, its expression reflects the cellular E2F activity. Transient
reporter assays indicate that the effects of epirubicin, its cellular targets p53 and
E2F1 are mediated through a proximal E2F-binding site within FOXM1 promoter (Fig.
4.6). In agreement, a recent study revealed that a great majority of genes repressed
by p53 and p73 contains E2F-binding sites, suggesting that p53 proteins repress
gene expression through inhibiting E2F activity (Scian, Carchman et al. 2008). The
direct binding of pRB and E2F1 on FOXM1 promoter was confirmed in vivo by ChIP
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analysis. ChIP assays also revealed that upon epirubicin treatment pRB level
increased and E2F1 level decreased within FOXM1 promoter region containing the
E2F-binding site (Fig. 4.4). Collectively, these findings indicate that epirubicin can
repress FOXM1 expression through induction of p53, which in turn represses E2F
activity through activating pRB and downregulating E2F1 expression.
4.4.2 p53 status is not a determinant of epirubicin response
Many chemotherapy agents in the treatment of cancer cause DNA damage that
is sensed by the tumour suppressor protein p53, triggering DNA repair and inducing
apoptosis (Liu and Kulesz-Martin 2001). In case of mutation or deletion of p53 gene,
the efficiency of chemotherapy agents is compromised (Aas, Børresen et al. 1996,
Reles, Wen et al. 2001). Mutations of p53 occur in more than half of all tumours and
have been linked to drug resistance (Hollstein, Sidransky et al. 1991). Despite that
the loss or mutation of p53 is associated with resistance to chemotherapy in many
cancers including breast cancer (Aas, Børresen et al. 1996), this study shows
evidence that DNA damage-sensing kinase ATM has also a role in regulating FOXM1
expression and epirubicin resistance, independent of p53. For instance, epirubicin
fails to activate p53 in MCF-7EPIR cells, but reduces FOXM1 protein expression in
combination with ATM inhibitor treatment (Fig. 4.11). Similarly, the combination of
epirubicin with ATM silencing completely abrogated FOXM1 protein expression (Fig.
4.12). Furthermore, U2OS cells treated with epirubicin activates p53 but only reduces
FOXM1 levels in combination with caffeine (an ATM inhibitor) (Millour, de Olano et al.
2011). Taken together, this study shows that p53 is not the major component of
epirubicin resistance. The role of p53 in drug sensitivity occurs through the activation
of cell cycle arrest and apoptosis genes transcription. Based on its role, the
transfection of p53 in MCF-7EPIR was tested, but p53 was not sufficient to restore
epirubicin sensitivity (data not shown). Similarly, ectopic expression of p53 in lung
cancer cell lines failed to alter the sensitivity of the cell line to the chemotherapeutic
agents adriamycin, taxol and carboplatin (Breen, Heenan et al. 2007).
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4.4.3 FOXM1 is a target of ATM
Based on the fact that ATM controls DNA repair through protein
phosphorylation leading to the activation of gene transcription, we analysed the
regulation of FOXM1 by ATM. Downregulation of ATM using siRNA significantly
reduced FOXM1 mRNA levels, indicating that ATM regulates FOXM1 at
transcriptional level (Fig. 4.14A). Consistently, ample evidence has demonstrated
that ATM regulates E2F1 expression in response to DNA damage, although the
mechanism involved is not completely understood (Blattner, Sparks et al. 1999,
Carcagno, Ogara et al. 2009). For example, genotoxic stress has been reported to
upregulate E2F1 expression at transcriptional level through the activation of ATM
(Carcagno, Ogara et al. 2009). On the contrary, a previous study also showed that
E2F1 expression is upregulated in response to DNA damage because of an increase
in protein stability indicating a post-translational regulation mechanism (Blattner,
Sparks et al. 1999). Current evidence indicates that E2F1 expression can be involved
in proliferation and tumorigenesis as well as apoptosis and tumour suppression
(Kusek, Greene et al. 2000, Polager and Ginsberg 2009). However, in the context of
cancer chemotherapy, the current observations evidently suggest that E2F1 is linked
to cell survival through promoting FOXM1 expression. In a previous microarray study,
E2F1-3 proteins have been shown to promote the expression of genes involved in
DNA replication, DNA repair and mitosis, and interestingly some of these E2F-
regulated genes identified, such as CYCLIN B1, are also transcriptional target of
FOXM1 (Polager, Kalma et al. 2002, Ren, Cam et al. 2002, Russo, Magro et al.
2006). Consistently, a number of recent studies have demonstrated that E2F1
expression is induced by a variety of DNA damaging agents and genotoxic
chemotherapeutic drugs and mirrors that of p53. Based upon our current findings that
ATM induces E2F activity and FOXM1 expression in response to DNA damage and
that E2F can promote FOXM1 transcription, this study proposes that ATM enhances
E2F1 expression and activates E2F-dependent FOXM1 expression at transcriptional
level in response to DNA damaging agents, such as epirubicin.
In addition, western blotting and immunoprecipitation experiments (Figure 4.13)
in MCF-7 and MCF-7EPIR cells suggested that FOXM1 phosphorylation is enhanced
by epirubicin in MCF-7EPIR cells. Taken together, our findings showed FOXM1
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involvement in DNA damage response pathway and for the first time a relationship
between FOXM1 and ATM. Numerous studies have shown that ATM regulates by
phosphorylation numerous genes involved in cell cycle checkpoints, apoptosis and
DNA repair. For instance, ATM phosphorylates the checkpoint kinase 2 (CHK2) on
threonine 68 in response to DNA damage. When activated, CHK2 inhibits CDC25C
phosphatase preventing entry into mitosis and allowing repair of the DNA. In addition,
it has previously been shown that FOXM1 protein is phosphorylated by CHK2 on
serine 361 in response to DNA damage. This phosphorylation event has also been
proposed to increase the stability of the FOXM1 protein to promote expression of
DNA repair genes (Tan, Raychaudhuri et al. 2007). Given that CHK2 functions
directly downstream of ATM in DNA damage response, it is predicted that the
induction of FOXM1 expression by ATM may therefore also occur through post-
translational mechanisms in response to DNA damage (Tan, Raychaudhuri et al.
2007). In contrast, silencing experiments showed that neither CHK2 nor CHK1 is
involved in FOXM1 regulation in MCF-7EPIR cells (Figure 4.13C). Given that FOXM1
is stabilised after epirubicin in MCF-7EPIR cells and that ATM knock-down completely
abolished FOXM1 expression, we hypothesised that ATM could directly
phosphorylate FOXM1. To investigate this hypothesise, I performed in vitro
radioactive-labelled kinase assay using ATM, FOXM1 and CHK2 (positive control)
recombinant proteins. While CHK2 protein did phosphorylate FOXM1 as already
published (Tan, Raychaudhuri et al. 2007), I did not observe any phosphorylation
events when ATM was added to FOXM1 protein (data not shown).
4.4.4 FOXM1 involvement in DNA repair and cell survival
Increased ATM expression in MCF-7EPIR cells indicates that ATM may promote
DNA repair to counteract the DNA damage-induced cell death triggered by genotoxic
chemotherapy drugs. Consistent with this, the sustained levels of foci in the MCF-
7EPIR cells after epirubicin are significantly reduced when compared with the drug
sensitive MCF-7 cells, as revealed by the P-H2AX staining (Fig.4.9). Moreover, this
idea is further supported by our finding that depletion of ATM activity (by siRNA or
Ku-55933 inhibitor) abolished the accumulation of FOXM1. Consequently, we
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showed that FOXM1 silencing reduced cell survival (Fig. 4.15), and could sensitise
the resistant MCF-7-EPIR cells to epirubicin sensitivity. Conversely, FOXM1
overexpression delays apoptosis induced by epirubicin (Fig. S.D.7.3) and by cisplatin
in MCF-7 cells as well as increased DNA repair (Kwok, Peck et al. 2010). Taken
together, this study shows FOXM1 involvement in ATM DNA damage response.
4.4.5 Conclusion
In summary, our data suggest that the genotoxic chemotherapy agent,
epirubicin, triggers the accumulation and activation of p53 and ATM, and it is the
antagonistic signals of activated ATM and p53 that converge on E2F to control
FOXM1 expression, DNA damage repair and cell survival. Specifically, p53 represses
while ATM enhances E2F activity, FOXM1 expression, cell survival in response to
epirubicin. In consequence, the development of epirubicin resistance can be due to
the loss of p53 function and an increase in ATM expression and activity. The finding
that ATM as well as p53 modulates FOXM1 expression may have important
implications for the diagnosis and treatment of drug resistant cancers, particularly
those lacking functional p53. For example, ATM and FOXM1 inhibitors can be
important cancer therapeutics as they can cause cell death independent of p53
status. ATM and FOXM1 inhibitors can also be used in combination with conventional
genotoxic therapeutic agents to enhance drug efficacy and overcome resistance.
Furthermore, p53, ATM and FOXM1 could be useful biomarkers for the prediction of
epirubicin sensitivity in cancer patients.
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Figure 4.16 Antagonistic pathways in epirubicin treatment. Epirubicin triggers the accumulation and activation of p53 and ATM. It is antagonistic signals of activated ATM and p53 that converge on E2F directly or indirectly to control FOXM1 expression and might regulate DNA damage repair and cell survival.
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4.5 Future work
Based on our results showing that epirubicin resistant MCF-7EPIR cells have
lost p53 activity and that DNA damage agents act through p53, the identification of
new therapeutic agents is needed in combination with DNA agents to resensitise
cells to cytotoxic drugs. DNA damage induces a cascade of protein kinases that
repair DNA breaks through the regulation of DNA repair proteins. Emerging
evidences show FOXM1 involvement in DNA repair response (Tan, Raychaudhuri et
al. 2007, Kwok, Peck et al. 2010). It was first reported that CHK2 phosphorylates
FOXM1 leading to its phosphorylation and regulation of DNA repair genes, XRCC1
and BRCA2 (Tan, Raychaudhuri et al. 2007). In our study, we showed a different
mechanism of FOXM1 regulation in which ATM regulates FOXM1 at transcriptional
level. Some evidence suggests that ATM also regulates FOXM1 at post-translational
level, but our attempts to perform in vitro kinase assay failed. It would be beneficial to
verify whether ATM can regulate FOXM1 phosphorylation and investigate the
phosphorylation site involved. An alternative would be that FOXM1 is phosphorylated
by another DNA damage kinase. Furthermore, investigation of the detailed role of
FOXM1 in DNA repair would clarify what is FOXM1 primary role in the epirubicin
resistant cells.
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CHAPTER 5 FOXM1 regulates ATM phosphorylation and DNA
damage response via transcriptional activation of NBS1
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5.1 Introduction
Breast cancer is the most cancer in women and one of the most prevalent
causes of death in women due to cancer relapse and metastasis to other organs
(Jemal, Siegel et al. 2009). Cytotoxic chemotherapeutic drugs are largely used in the
treatment of many cancers from different origins and to reduce the chance of
metastasis (Martin, Villar et al. 2003, Smith and Chua 2006). Chemotherapy agents
encompass alkylating agents, anti-metabolite, topoisomerase inhibitors,
anthracyclines and anti-mitotic agents (Hortobagyi 1995, Rodler, Korde et al. 2010,
Rodríguez-Lescure 2010). Each class of drug act differently, but all lead to DNA
damages. Among cytotoxic chemotherapies, anthracyclines are anti-cancer
antibiotics widely used and effective for the treatment of breast, lung, ovarian and
leukaemia cancers (Lown 1993). Anthracyclines mechanism of action is thought to
interfere with enzymes involved in DNA replication, but is also likely to be involved in
DNA intercalation and DNA damage (Euhus 2011). ATM is a key factor activated
following DNA damage that induces phosphorylation of its downstream target histone
H2AX, leading to the recruitment of DNA repair proteins to the sites of damage
(Fernandez-Capetillo, Chen et al. 2002, Celeste, Fernandez-Capetillo et al. 2003).
Although ATM signalling pathway and downstream targets are known, further
elucidations are necessary to understand all mechanisms activating ATM and DNA
repair. It has been reported that changes in chromatin structure induced by the
MRE11/RAD50/NBS1 (MRN) complex was essential for ATM auto-phosphorylation
and activation (Lee and Paull 2004, Lee and Paull 2005).
The mammalian FOXM1 transcription factor belongs to the forkhead box
superfamily and plays a critical role in cell proliferation, as it is required for G1/S and
G2/M cell cycle transitions (Laoukili, Kooistra et al. 2005, Wierstra and Alves 2007).
Besides its role in cell growth, FOXM1 also regulates organogenesis, angiogenesis,
metastasis and DNA damage repair (Dai, Kang et al. 2007, Tan, Raychaudhuri et al.
2007, Raychaudhuri and Park 2011). Consistent with its roles, FOXM1 is found
elevated in a broad spectrum of carcinomas (Kalinichenko, Major et al. 2004,
Pilarsky, Wenzig et al. 2004, Chandran, Ma et al. 2007, Zeng, Wang et al. 2009). In
addition to its involvement in tumorigenesis, FOXM1 dysregulation was implicated in
tamoxifen resistant breast cancer cells through abrogation of tamoxifen anti-
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proliferative effect (Millour, Constantinidou et al. 2010). Furthermore, FOXM1
dysregulation has been shown to play a role in the development of cisplatin and
epirubicin resistance in breast cancer (Kwok, Peck et al. 2010, Millour,
Constantinidou et al. 2010, Millour, de Olano et al. 2011). The role of FOXM1 in
resistance to DNA damage agents is thought to be due to enhanced DNA repair.
However, FOXM1 involvement in DNA damage signalling pathway has not been
explored. In this report, the role of FOXM1 in ATM DNA damage response in
epirubicin resistance was studied.
5.2 FOXM1 is involved in single and double stranded DNA
repair in epirubicin resistant breast cancer cells
The previous study chapter 4 and published work from our laboratory showed
FOXM1 involvement in resistance to DNA damage agents including epirubicin and
cisplatin. The study chapter 4 showed that epirubicin enhances P-H2AX DNA
damage foci in epirubicin sensitive breast cancer cells, while P-H2AX foci are
sustained and low in epirubicin resistant breast cancer cells. This study suggests a
higher DNA repair system in the epirubicin resistant cell line (Millour, de Olano et al.
2011). To investigate single stranded DNA repair mechanism of epirubicin resistant
MCF-7EPIR cells, the host-cell reactivation assay (HCR) was used. The plasmid
harbouring firefly luciferase was damaged by a nicking endonuclease on a single
strand of the DNA and repaired by the cellular DNA repair machinery. Only fully
repaired plasmid transcribed correctly generate active firefly luciferase (Fig. 5.1)
(Matijasevic, Precopio et al. 2001). According to differences in transfection
efficiencies, the luciferase data were not normalised to the undamaged control
plasmid, but normalised to Renilla and compared to the time 0 h (from the same
transfection mix). Additionally, all controls with undamaged plasmid were performed
(data not shown). The firefly luciferase activity of the damaged plasmid was
recovered by 1174-fold 72 h post-transfection in epirubicin resistant MCF-7EPIR cells,
while luciferase activity was only recovered by 5.2-fold 72 h after transfection in
epirubicin sensitive MCF-7 cells (Fig. 5.1A). This result indicates that MCF-7EPIR
cells have an enhanced mechanism of repair for single strand damage than MCF-7
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cells. FOXM1 was examined for its role in DNA repair in MCF-7EPIR cells using the
HCR assay. The firefly luciferase activity of the damaged plasmid transfected
combined with the non-specific siRNA (NS siRNA) in MCF-7EPIR cells was recovered
by 24.5-fold after 48 h transfection, which matches the result obtained Figure 5.1A
(Fig. 5.1B). In contrast, FOXM1 silencing (FOXM1 siRNA) completely abrogated the
luciferase recovery, suggesting that FOXM1 plays an important role in DNA repair in
MCF-7EPIR cells (Fig. 5.1B. Notably, these results remain preliminary and manual. It
has also been reported that dysregulated FOXM1 is involved in cisplatin and
epirubicin resistance and it is thought to be due to enhanced DNA repair
mechanisms. Our lab has recently investigated FOXM1´s role in double stranded
DNA repair using HeLa cell lines harbouring an integrated direct repeat green
fluorescent protein reporter for HR or NHEJ. These experiments showed that FOXM1
depletion reduced the HR DSB repair, but had no significant effects on NHEJ repair
(Monteiro, Khongkow et al. 2012).
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Figure 5.1 FOXM1 depletion alters single stranded DNA repair in MCF-7EPIR
cells. A.
MCF-7EPIR
and MCF-7 cells were transiently co-transfected with damaged firefly luciferase and undamaged renilla luciferase plasmids. After 24 h transfection, luciferase activities were assayed at 0, 48 and 72 h and the ratios firefly/renilla were calculated. Folds increase
relative to 0 h are shown. B. MCF-7EPIR
cells were either transfected with non-specific (NS) siRNA or FOXM1-targeting siRNA (100 nmol/L). Twenty-four hours after transfection, cells were co-transfected with damaged firefly luciferase and undamaged renilla luciferase plasmids, and luciferase activities were assayed at 0, 24 and 48 h and the ratios firefly/renilla were calculated. Folds increase relative to 0 h are shown. Columns, means derived from three independent experiments; bars, SD.
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5.3 Enhanced recruitment of FOXM1 and P-H2AX in MCF-
7EPIR cells following DNA breaks
As MCF-7EPIR cells have high DNA repair in response to epirubicin (Fig. 4.9
and 4.10), the recruitment of DNA repair proteins at double strand breaks (DSB) sites
was investigated. For this purpose, the eukaryotic homing endonuclease I-Ppol,
which has a 15 base pair recognition sequence to cleave endogenous DNA sites in
the human genome, was used to cleave specifically one site on the chromosome 1
on an intron of the DAB1 gene. Expression of the I-Ppol leads to a cleavage of the I-
Ppol target sites (DAB1 gene), generating DSBs (equivalent to 0.8 Gy irradiation) and
activating ATM-dependent signalling pathway (Berkovich, Monnat et al. 2007).
Although it is inaccurate to compare DNA damage epirubicin-induced with DNA
damage I-Ppol-induced because of the difference in DNA damage levels, the
generation of DSBs by I-Ppol transfection was confirmed in MCF-7 and MCF-7EPIR
cells using the expression level of P-H2AX (Fig. 5.2). Western blotting of I-Ppol would
have also been a good control in this case. The P-H2AX and FOXM1 were
immunoprecipitated and the DNA sequence bound to these proteins was amplified
using DAB1 primers and normalised with -actin housekeeping gene. DNA breaks
induced by I-Ppol transfection induced a strong enhancement in the recruitment of P-
H2AX and FOXM1 proteins on the sites of DNA breaks in MCF-7EPIR cells relative to
MCF-7 cells and to the respective IgG negative control (Fig. 5.2). These results
suggest that the recruitment of DNA repair proteins to the DSB sites is enhanced in
MCF-7EPIR cells compared to MCF-7 cells epirubicin sensitive. In addition, these
data might indicate that FOXM1 may be necessary either for H2AX phosphorylation
or for the recruitment of P-H2AX at the DSBs. Impaired H2AX phosphorylation or
recruitment would affect DNA repair proteins recruitment.
However, these results remain preliminary and imprecise. Indeed, Michael
Kastan et al. improved this system by adding a mutant oestrogen receptor hormone-
binding domain to I-Ppol to create a fusion protein that localized to the nucleus in
response to 4-hydroxytamoxifen (4-OHT). Addition of 4-OHT to cells infected with an
oestrogen receptor-I-Ppol retrovirus results in a time-dependent cleavage of I-Ppol
site (Berkovich, Monnat et al. 2007). In Figure 5.2, the kinetic of transfection is likely
159
to be very different as MCF-7 cells are much easier and quicker to transfect than
MCF-7EPIR cells. Therefore, the level of H2AX recruitment in MCF-7 cells might be
low because the transfection is rapid in these cells and 24 hrs after transfection the
damages have already been repaired. Western blotting of I-Ppol in a time course
could also provide us with extra information about the kinetic of transfection.
However, the difference between the level of P-H2AX in the western blot and ChIP
experiments for MCF-7 cells is striking. H2AX recruitment on DSB might not be on
the amplified site. H2AX protein recruitment might also be low because these MCF-7
cells have a low level of DNA damage repair pathway activation. Indeed, in Figure
4.12 MCF-7 cells showed a very low level or no phospho-ATM after epirubicin
treatment. The experiment has been repeated several times using western blot with
high sensitivity detection methods, but the expression of phospho-ATM remained
undetectable.
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Figure 5.2 Increased in the recruitment of FOXM1 and repair factors at DNA breaks in
MCF-7EPIR
cells. A. MCF-7 and MCF-7EPIR cells were transfected with with 3 μg of empty vector or vector encoding for I-Ppol and harvested for western blot analysis 48 h post-transfection. The protein expression levels were determined for P-H2AX and β-tubulin. B. Transient expression of the I-Ppol for 24 h leads to a cleavage of the I-Ppol target sites and
generation of DSBs. After cross link reversal of epirubicin-treated MCF-7 and MCF-7EPIR
cells, the H2AXpSer139 and FOXM1 were immunoprecipitated and the DNA sequences bound to these proteins were amplified using DAB1 primers. Input DNA was used to normalise the amplified DNA. Columns, means derived from three independent experiments; bars, SD. Statistical analyses were done using Student’s t test. **, P≤0.01 and ***, P≤0.001, significant and n.s, non significant.
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5.4 FOXM1 is required for the activation of ATM, H2AX and
CHK2 DNA repair proteins
Since the HCR assay showed that FOXM1 is essential to repair DNA in MCF-
7EPIR cells and might be necessary for H2AX phosphorylation, the effect of FOXM1
silencing was examined on the phosphorylation of DNA repair proteins. For this
purpose, MCF-7EPIR cells were transiently transfected with non-specific siRNA and
FOXM1-targeting siRNA and examined for their ability to phosphorylate ATM and its
targets when treated with 1 μmol/L of epirubicin. Western blot analysis confirmed that
FOXM1 was effectively silenced at least over 48 h after treatment (Fig. 5.3A). The
use of phosphospecific antibodies showed an increase in the phosphorylation of
ATM, H2AX and CHK2 following epirubicin exposure in the non-specific siRNA
condition (Fig. 5.3A). In contrast, the phosphorylation of ATM was completely
abrogated, and phosphorylations of H2AX and CHK2 were reduced in treated MCF-
7EPIR cells when FOXM1 was silenced (Fig. 5.3A). To further confirm the reduction
in ATM phosphorylation, the percentage of P-ATM positive cells were assessed by
staining using flow cytometry in MCF-7EPIR cells treated with non-specific and
FOXM1-targeting siRNA. After 48 h treatment with 1 μmol/L of epirubicin, the
percentage of P-ATM positive cells increased compared to untreated condition in the
non-specific siRNA condition, while it decreased in cells treated with siRNA targeting
FOXM1, independent of epirubicin treatment (Fig. 5.3B). The validation of FOXM1
silencing was also confirmed by staining using flow cytometry, and for each antibody
an IgG secondary antibody negative control was performed (Fig. 5.3B). Notably,
FOXM1 enhancement at protein level Figure 5.3A was not observed using flow
cytometry indicating that total FOXM1 protein expression remained the same and
that FOXM1 induction observed by western blotting could be due to an increase in its
phosphorylation levels. The reduction in H2AX phosphorylation observed Figure 5.3A
was confirmed by immunofluorescence staining in MCF-7EPIR cells treated with non-
specific and FOXM1-targeting siRNA in absence and presence of epirubicin at 1
μmol/L for 24 h (Fig. 5.4). Although our previous study chapter 4 showed low levels
of P-H2AX foci in epirubicin resistant cells compared to MCF-7 cells, the foci were
present and sustained over the treatment (Millour, de Olano et al. 2011). In this
study, the quantification of P-H2AX foci in MCF-7EPIR cells showed a decrease of
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foci in cells treated with siRNA targeting FOXM1 compared to non-specific siRNA
condition at 24 h epirubicin treatment (Fig. 5.4B). This result is in opposition to results
from a recent study using the same cell lines (Monteiro, Khongkow et al. 2012).
However, these results were confirmed in a human fibroblast cell line (Fig. 5.5).
FOXM1 silencing in human fibroblast cells also abrogated ATM, H2AX and Chk2
phosphorylations, and by consequent their activation (Fig. 5.5 and 5.6). Figure 5.5
the use of the siRNA targeting ATM should be used to confirm the specificity of the
phosphor-ATM antibody and total Chk2 and H2AX antibodies should be used Figure
5.6 as control. Taken together, these data show that FOXM1 silencing reduced
phosphorylation events following DNA damage response in MCF-7EPIR cells and
suggest that it may reduce recruitment of these proteins at the DSBs sites.
Figure 5.3 FOXM1 silencing reduces ATM phosphorylation on serine 1981 in MCF-
7EPIR
cells. A. MCF-7EPIR
cells were either transfected with non-specific (NS) siRNA or
FOXM1-targeting siRNA (100 nmol/L). Twenty-four hours after transfection, MCF-7EPIR
cells were treated with 1 µmol/L of epirubicin and harvested for western blot analysis at 0, 24 and 48 h. The protein expression levels were determined for FOXM1, ATMpSer1981, ATM,
H2AXpSer139, H2AX, Chk2pThr68, Chk2 and β-tubulin. B. MCF-7EPIR
cells transfected with siRNA NS and siRNA FOXM1 untreated and treated with 1 µmol/L of epirubicin 48 h were assessed by staining with an antibody against ATMpSer1981, or FOXM1, or an isotype IgG (negative control), followed by an Alexa 488-conjuguated secondary antibody. The percentage of cells ATMpSer1981 or FOXM1 positive were determined by flow cytometry. Columns, means derived from three independent experiments; bars, SD. Statistical analyses
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were done using Student’s t test. **, P≤0.01 and ***, P≤0.001, significant and n.s, non significant.
Figure 5.4 FOXM1 silencing decreases H2AX phosphorylation on serine 139 in MCF-
7EPIR
cells. A. MCF-7EPIR
cells were either transfected with non-specific (NS) siRNA or
FOXM1-targeting siRNA (100 nmol/L). Twenty-four hours after transfection, MCF-7EPIR
cells were treated with 1 µmol/L of epirubicin for 24 h and stained with H2AXpSer139 antibody (green) and DAPI (red). Images visualized by confocal microscopy. Images: magnification: x 20; insets x 80. B. The results were quantified using Image J and were the average of three independent experiments. Columns, means derived from three independent experiments; bars, SD. Statistical analyses were done using Student’s t test. **, P≤0.01 and ***, P≤0.001, significant and n.s, non significant.
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Figure 5.5 FOXM1 silencing in human fibroblast cells abrogates ATM phosphorylation. Human fibroblast (48BRhtert) cells were either transfected with non-specific (NS) siRNA or FOXM1-targeting siRNA (100 nmol/L). Twenty-four hours after transfection, cells were treated with 1µmol/L of epirubicin and harvested for western blot analysis at 0, 24 and 48 h. The protein expression levels were determined for FOXM1, ATMpSer1981, ATM, FOXM1 and β-tubulin.
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Figure 5.6 FOXM1 silencing in human fibroblast cells decreases Chk2 and H2AX phosphorylation. Human fibroblast (48BRhtert) cells were cultured, treated with 1 μmol/L of epirubicin for 48 h and stained with Chk2pThr68 and H2AXpSer139 antibodies (red) and DAPI (blue). Images visualized by confocal microscopy. Images: magnification: x 20; insets x 80.
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5.5 FOXM1 is required for ATM auto-phosphorylation upon
epirubicin
It has been reported that ATM is regulated by the members of E2F family either
at post-translational level or at promoter level (Berkovich and Ginsberg 2003, Hong,
Paulson et al. 2008). To elucidate the mechanism by which FOXM1 regulates ATM,
ATM mRNA level was examined in MCF-7EPIR cells when FOXM1 was silenced in
combination with 1 μmol/L of epirubicin. RT-qPCR analysis showed that FOXM1
silencing did not affect ATM mRNA levels compared to the non-specific siRNA
condition in MCF-7EPIR cells (Fig. 5.7A). Furthermore, western blot analysis of wild-
type (wt) and FOXM1 knock-out (Foxm1-/-) MEFs cells treated with 1 μmol/L
epirubicin showed a reduction in P-ATM level, while ATM mRNA levels remained
steady in Foxm1-/- cells (Fig. 5.7B and C). Taken these results together, the reduction
of FOXM1 levels by transient knock-down or genomic knock-out decreased the level
of ATM phosphorylation, but did not affect its mRNA levels. The reverse was showed
with FOXM1 overexpression in wt MEFs that increased ATM phosphorylation as well
as ΔN-FOXM1 overexpression (Fig. 5.7D). Collectively, these data indicate that
FOXM1 plays a role in regulating ATM auto-phosphorylation on serine 1981.
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Figure 5.7 FOXM1 does not regulate ATM transcriptionally. A. MCF-7EPIR
cells were either transfected with non-specific (NS) siRNA or FOXM1-targeting siRNA (100 nmol/L).
Twenty-four hours after transfection, MCF-7EPIR
cells were treated with 1 µmol/L of epirubicin and harvested for RT-qPCR analysis at 0 and 24 h. ATM mRNA levels were determined and normalised to L19 gene. MEFs wild-type (WT) and knock-out for FOXM1 (Foxm1-/-) were treated with 1 µmol/L of epirubicin and harvested at indicated times for western blot (B.) and RT-qPCR analyses (C.). The protein expression of ATMpSer1981, ATM, FOMX1 and β-tubulin and the ATM mRNA were determined. Columns, means derived from three independent experiments; bars, SD. D. Wild-type MEFs were transiently transfected with empty vector and vectors encoding for FOXM1 and ΔN-FOXM1 and harvested after 24 h for western blot analysis. The protein expression of ATMpSer1981, ATM, FOXM1 and β-tubulin were examined.
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5.6 Transcriptional regulation of NBS1 by FOXM1
It has been reported that ATM is recruited and fully activated at the DSB sites
by the MRN complex (Lee and Paull 2004, Lee and Paull 2005). The modulation of
ATM phosphorylation via the regulation of the MRN complex through FOXM1 was
investigated in MCF-7EPIR cells. To investigate whether FOXM1 regulates members
of the MRN complex, the effect of FOXM1 silencing was examined on MRE11,
RAD50 and NBS1 mRNA levels in MCF-7EPIR cells. RT-qPCR data revealed that
FOXM1 knock-down reduced significantly NBS1 mRNA, but has no effect on MRE11
and RAD50 mRNA levels in MCF-7EPIR cells (Fig. 5.8A). This finding was confirmed
in the human fibroblast cell line (Fig. 5.8B). Indeed, FOXM1 silencing significantly
reduced NBS1 mRNA levels, but not MRE11 or RAD50 mRNA levels. This study was
extended to breast cancer cell lines including MDA-MB-231, ZR-75-1 and MCF-7
cells. Each of these cell lines showed a significant reduction of NBS1 mRNA after
FOXM1 silencing, indicating NBS1 as a potential transcriptional target of FOXM1
(Fig. 5.8C). Transient reporter assay was performed to study whether FOXM1
regulates NBS1 at promoter level. Full length FOXM1 did not affect significantly
NBS1 promoter activity (data not shown), but ectopic expression of the active
FOXM1 form, ∆N-FOXM1, induced a significant increase in the luciferase activity of
NBS1 promoter (WT FHK-luc) in MCF-7 cells in a dose-dependent manner (Fig.
5.9B). The study of NBS1 promoter revealed a forkhead binding site located at -78 pb
from the transcription start explaining the responsiveness of NBS1 promoter to
FOXM1 active form (Fig. 5.9A). Directed mutagenesis of the forkhead site (mFHK-
luc) abrogated the transcriptional induction of NBS1 promoter by ∆N-FOXM1
expression vector compared to the wild-type promoter WT FHK-luc (Fig. 5.9B).
Chromatin immunoprecipitation assays showed that FOXM1 binds NBS1 promoter
after ectopic expression of FOXM1 in MCF-7 cells and after epirubicin treatment in
MCF-7EPIR cells (Fig. 5.9C). These results suggest that FOXM1 could affect ATM
auto-phosphorylation via the transcriptional regulation of NBS1.
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Figure 5.8 FOXM1 silencing significantly decreases NBS1 mRNA levels. A. MCF-7EPIR
cells, B. Human fibroblast cells, and C. MDA-MB-231, ZR-75-1 and MCF-7 cells were transiently transfected with non-targeting siRNA and siRNA against FOXM1, and harvested for RT-qPCR analysis. The mRNA levels of FOXM1, NBS1, MRE11 and RAD50 were examined. Columns, means derived from three independent experiments; bars, SD. Statistical analyses were done using Student’s t test. *, P≤0.1 **, P≤0.01 and ***, P≤0.001, significant.
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Figure 5.9 FOXM1 binds directly to NBS1 promoter through the Forkhead binding site (FHK). A. Schematic representaiton of NBS1 luciferase promoter construction pXL2-Nbs1, wild-type Forkhead (WT FHK) and mutant Forkhead (mFHK) binding sites performed using site directed mutagenesis. B. HEK293T cells were cultured in 10% FCS DMEM medium and transiently transfected with pXL2-Nbs1 and increasing amount of deltaN-FOXM1 constructions (0 and 10 ng) and assessed for luciferase assay. All relative luciferase activity values are corrected for co-transfected Renilla activity. Columns, means derived from three independent experiments; bars, SD. Statistical analyses were done using Student’s t test. **, P≤0.01 and ***, P≤0.001, significant and n.s, non significant. C. Chromatin immunoprecipitation (ChIP) analysis of NBS1 promoter. MCF-7 cells were transfected with
pcDNA3 ( - ) or pcDNA3/FOXM1 ( + ) and MCF-7EPIR
cells were treated with epirubicin. These cells were used for ChIP assay using anti-IgG and anti-FOXM1 antibodies as indicated. After cross-linkinf reversal, the co-immunoprecipitated DNA was amplified by PCR using primers for NBS1 FHK containing region ( -119 to -1 pb ) and a control region ( -281 to -128 pb ) and runned in 1% agarose gel.
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5.7 NBS1 mediates ATM activation upon epirubicin
Since this study indicates that FOXM1 regulates NBS1 transcriptionally and
affects the phosphorylation of ATM, I hypothesised that FOXM1 regulates
transcriptionally NBS1, which in turn regulates the auto-phosphorylation of ATM at
the DNA break sites. To demonstrate that NBS1 is required for ATM activation, we
used the wt and Nbs1-/- MEFs cells transfected with empty control vector and vector
encoding for NBS1. The full activation of ATM is only observed in MEFs cells treated
with epirubicin and transfected with NBS1, but not in MEFs cells lacking NBS1 (Fig.
5.10). Taken together, these data indicate that ATM activation requires NBS1 and
epirubicin treatment. This finding suggests that FOXM1 may acts upstream of ATM
and NBS1 and could control ATM auto-phosphorylation through NBS1.
To determine whether FOXM1 can activate ATM auto-phosphorylation, western
blot analysis could be performed on Foxm1-/- cells transfected with empty vector or a
plasmid encoding for NBS1 to circumvent the lack of FOXM1 and activate ATM.
Figure 5.10 NBS1 is required for ATM activation upon epirubicin. NBS-LBI cells were either transfected with 3 μg of empty vector or vector encoding for NBS1 and treated with 1 µmol/L of epirubicin 24 h and harvested for western blot analysis at 0, 24 and 48 h. The protein expression levels were determined for ATMpSer1981, ATM, FOXM1, Chk2, NBS1 and β-tubulin.
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5.8 Discussion
Chemotherapeutic drugs generate double strand breaks by free radical attack of
deoxyribose, inhibition of re-ligation of DNA strand broken by topoisomerase II or
DNA replication. DNA DSBs are the most deleterious form of DNA damage because
they do not leave an intact complementary strand to be used as a template for DNA
repair and they induce DNA damage signalling pathway leading to cell cycle arrest,
apoptosis or repair. Deregulation in DNA damage signalling pathway can favour DNA
repair and inhibit apoptosis to contribute to chemotherapeutic drug resistance. Cells
rely on two major DNA DSBs repair pathways: homologous recombination (HR) and
non-homologous end-joining (NHEJ). HR requires a homologous template, sister
chromatid, and allows repair of DSBs in S and G2 phases of the cell cycle (San
Filippo, Sung et al. 2008, Moynahan and Jasin 2010). In contrast, NHEJ can operate
throughout the cell cycle without the need for DNA template.
5.8.1 FOXM1 is involved in ATM and its downstream
substrates phosphorylations
In response to DSBs, DNA damage signalling pathway delays the cell cycle
before and during DNA replication (G1/S and intra-S checkpoints) and before cell
division (G2/M checkpoints) to prevent duplication and segregation of damaged DNA.
DNA damage signalling cascades are complex events that require various proteins
whose function can be categorised as DNA damage sensors, transducers, mediators
and effectors. DNA damage signalling pathway involves two key serine/threonine
kinases: ATM (ataxia telangiectasia mutated) and ATR (ATM and rad3-related). The
MRE11/RAD50/NBS1 sensor complex detects DSBs and contributes to the
recruitment and activation of the ATM transducer. Mediator proteins, such as MDC1
(mediator of DNA damage checkpoint), 53BP1 (p53-binding protein 1) and BRCA1,
help activate effector kinases CHK1 and CHK2, which spread the signal throughout
the nucleus (Lavin, Delia et al. 2006). In this study, FOXM1 silencing abrogates ATM
phosphorylation in MCF-7EPIR and fibroblast cells, which is required for
phosphorylation events of the DNA damage cascade (Fig. 5.3A and 5.5). Similarly,
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the level of P-ATM positive cells was reduced when cells were transfected with
FOXM1-targeting siRNA (Fig. 5.3B). FOXM1 silencing also reduces phosphorylation
of CHK2 and P-H2AX in MCF-7EPIR cells (Fig. 5.3A) and human fibroblasts (Fig.
5.6), which inhibits activation of transcription factors, cell cycle and apoptosis
regulators and repair proteins (Matsuoka, Rotman et al. 2000, Bartek and Lukas
2003, Iliakis, Wang et al. 2003). Although these results have been confirmed in
different cell lines, it is in contradiction with results recently published. In this study I
suggest that FOXM1 inhibition reduces P-H2AX and the activation of the DNA repair
pathway, while the recent study shows that FOXM1 depletion increases P-H2AX and
DNA damage. H2AX is a histone protein that is rapidly phosphorylated by ATM in
response to DNA damage. Activated H2AX forms foci around the DSBs and helps to
recruit the proteins responsible for DNA repair. In this study, I hoped to show that
FOXM1 silencing reduced phosphorylations and recruitment of DNA repair proteins
at the DSBs.
The mechanism of ATM regulation by FOXM1 was investigated by western blot
and RT-qPCR. The results revealed that FOXM1 silencing inhibits ATM auto-
phosphorylation, but does not affect total ATM protein and mRNA levels (Fig. 5.7). A
recent study demonstrates that FOXO3A interacts with ATM to promote its auto-
phosphorylation on serine 1981 (Tsai, Chung et al. 2008). However, ATM and
FOXM1 direct interaction was not found (data not shown), suggesting that FOXM1
activates ATM auto-phosphorylation indirectly.
5.8.2 FOXM1 regulates NBS1
The role of the MRN complex in ATM activation has clearly been shown through
analysis of ATM-dependent phosphorylation events in cells with MRN deficiencies
(Uziel, Lerenthal et al. 2003). MRN complex stimulates ATM activation that induces
p53, CHK2 and H2AX in vitro phosphorylations using recombinant proteins. The
association between ATM and MRN is mediated through multiple protein-protein
interactions, one between ATM and NBS1, and the other between ATM and
MRE11/RAD50 (Lee and Paull 2004, You, Chahwan et al. 2005). As FOXM1 is a
transcription factor, the transcriptional regulation of the MRN protein complex by
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FOXM1 was investigated. RNA interference experiments demonstrated that FOXM1
knock-down significantly decreased NBS1 mRNA in different human breast cancer
cell lines and fibroblasts (Fig. 5.8). Furthermore, ectopic expression of the active form
of FOXM1 induced NBS1 promoter activity, while mutation of the forkhead binding
site abrogated NBS1 promoter activity induction following addition of FOXM1 active
form (Fig. 5.9). Chromatin immunoprecipitation assays showed that FOXM1 directly
binds NBS1 promoter following FOXM1 overexpression and DNA damage treatment
(Fig. 5.9C). Similarly, NBS1 is a target of a transcription factor involved in cell growth
control, C-MYC (Chiang, Teng et al. 2003). C-MYC function was known in promoting
cell proliferation in normal and neoplastic cells, until its function was linked to the
regulation of DNA DSB repair pathway.
5.8.3 NBS1 activates ATM auto-phosphorylation
Even though wild-type ATM is present, only reconstitution of MRN complex
restores ATM auto-phosphorylation and phosphorylation of downstream substrates in
MRN deficient cells (Uziel, Lerenthal et al. 2003, Lee and Paull 2004). In this study,
rescue experiments in Nbs1-/- fibroblast cells also showed that the combination of
epirubicin treatment with NBS1 fully induces ATM auto-phosphorylation (Fig. 5.10).
Given that our promoter assay showed that FOXM1 regulates NBS1, the results
could suggest that FOXM1 activates ATM through NBS1 transcription (Fig. 5.11).
Similarly, NBS1 is required for E2F1 to induce p53 phosphorylation. In fibroblasts
lacking NBS1, p53 and CHK2 phosphorylations were impaired, while E2F1 induced
p53 and CHK2 phosphorylations in wild-type cells (Powers, Hong et al. 2004).
5.8.4 FOXM1 function in DNA repair
The role of FOXM1 in DNA repair was first showed in osteosarcoma cells with
the direct regulation of XRCC1 and BRCA2, genes involved in DNA repair (Tan,
Raychaudhuri et al. 2007). In this study, I showed that FOXM1 regulates NSB1, a
sensor of DNA damage. A study found NBS1 as a downstream target of C-MYC
proliferation factor and showed the role of C-MYC in DNA repair for the first time
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(Chiang, Teng et al. 2003). This study is also the first evidence of FOXM1 function in
ATM activation and ATM-mediated DNA damage phosphorylation cascade. DNA
damage signalling pathway activates DNA damage sensors, transducers, mediators
and effectors required for cell cycle arrest, apoptosis and DNA repair. Cells were co-
transfected with pre-damaged single strand DNA template and undamaged Renilla.
The level of repair of damaged template was significantly enhanced in MCF7-EPIR
cells relative to MCF-7 cells, whereas DNA repair was prevented in MCF7-EPIR cells
treated with siRNA FOXM1 relative to non-specific siRNA (Fig. 5.1). These results
indicate that FOXM1 is involved in HR. Furthermore, a recent study demonstrated the
role of FOXM1 in HR, but not in NHEJ (Monteiro, Khongkow et al. 2012).
A growing body of evidence indicate the importance of chromatin organisation
in the DNA damage response, with the most prominent modifications being the
phosphorylation of histone H2AX at the site of DNA breaks. As H2AX is associated
with chromatin at DNA break sites and plays a key role in recruiting DNA repair
proteins to nuclear foci, the recruitment of FOXM1 to DNA break sites was assessed
by chromatin immunoprecipitation upon local induction of DNA damage by specific
endonucleases. The generation of sequence-specific DSBs by I-Ppol endonuclease
(Monnat, Hackmann et al. 1999, Berkovich, Monnat et al. 2008) was verified by
H2AX phosphorylation (Fig. 5.2A). Transient transfection of I-Ppol in MCF-7EPIR cells
increased the recruitment of phosphorylated-H2AX at DNA breaks sites (Fig. 5.2B).
Importantly, DSBs enhance FOXM1 recruitment at DNA breaks in MCF-7EPIR cells
(Fig. 5.2B). In contrast, P-H2AX and FOXM1 recruitments at DNA breaks is not
increased following I-Ppol transfection in MCF-7 cells (Fig. 5.2A). This result is in
opposition with the result published by Berkovich et al. in which P-H2AX was
recruited on DSBs in MCF-7 cells after 16 hrs transfection with I-Ppol (Berkovich,
Monnat et al. 2007). Our MC-7 cell lines showed a low level of DNA repair as
observed Figure 5.1A. Hence, this experiment should be repeated with low passage
MCF-7 cell lines. This study did show a higher level of FOXM1 recruitment at DNA
breaks I-Ppol-induced as well as P-H2AX in MCF-7EPIR cells compared to MCF-7
cells (Fig. 5.2). H2AX interacts with MDC1, 53BP1, NBS1, RAD51 and BRCA1 at
DNA break sites, but because FOXM1 silencing decreased H2AX phosphorylation,
FOXM1 might affect protein assembly of sensors, mediators and effectors.
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5.8.5 Conclusion
This study shows that FOXM1 activates the transcription of NBS1, member of
the MRN sensor complex, inducing ATM auto-phosphorylation (on serine 1981) and
the phosphorylation of ATM downstream targets including H2AX and CHK2 in MCF-
7EPIR cells (Fig. 5.3 and 5.4) and in human fibroblasts (Fig. 5.5 and 5.6). These
results also showed that FOXM1 is required for the DNA damage phosphorylation
events, suggesting that FOXM1 might affect DNA repair mechanisms in MCF-7EPIR
cells. FOXM1 silencing completely abrogated the elevated DNA repair mechanism in
MCF-7EPIR cells (Fig. 5.1B). The examination of protein recruitment at DNA breaks
shows that FOXM1 is recruited after DNA breaks in MCF-7EPIR cells and might have
a role in protein assembly ATM-mediated (Fig. 5.2). Together these results indicate
that FOXM1 is required for DNA damage signalling and DNA repair, and involved in
protein assembly at DSB breaks. This study showed differences in DNA damage
signalling induction, DNA repair factor recruitment and DNA repair between breast
cancer cell sensitive and resistant to epirubicin (Fig. 5.11). Taken together, this study
unravels that FOXM1 has a crucial role in promoting DNA repair response in MCF-
7EPIR cells and suggests that targeting FOXM1 could potentially resensitise
epirubicin-resistant cells to DNA damage and cell death.
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Figure 5.11 Differential pathways upon epirubicin in sensitive and resistant MCF-7 cells. In epirubicin sensitive MCF-7 cells, epirubicin activates p53 which downregulates E2F to repress FOXM1 expression and arrest the cell cycle. In epirubicin resistant MCF-7 cells, epirubicin activates ATM which in turn activates its downstream targets H2AX, CHK2 and E2F involved in DNA repair. E2F positively activates FOXM1 expression, which regulates NBS1 at promoter level. NBS1 is required to activate ATM auto-phosphorylation, which creates a feedback loop, controlling DNA damage repair and survival.
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5.9 Future work
This report shows that targeting FOXM1 in epirubicin resistant breast cancer
cells is an attractive strategy considering that FOXM1 is overexpressed in these cells
and that FOXM1 inhibition results in DNA repair defect in these cells. Epirubicin
resistant breast cancer cells have a high capacity to repair DNA, while FOXM1
silencing reduces ATM and its downstream targets activation. A previous study
showed that FOXM1 regulates XRCC1 and BRCA2 DNA repair genes, consistent
with FOXM1 inhibition resulting in defective DNA repair (Tan, Raychaudhuri et al.
2007). XRCC1 is important for two types of DNA repair, HR and NHEJ, while several
lines of evidence indicate that BRCA genes are only critical for DSB repair by HR. In
addition, this study showed that FOXM1 regulates NBS1 that is involved in both DSB
repair mechanisms. Furthermore, a recent study showed that FOXm1 is involved in
HR, but not in NHEJ (Monteiro, Khongkow et al. 2012). HR is particularly important
during S and G2 phases, while NHEJ is dominant during G0, G1 and early S phases.
It would be interesting to investigate whether a chemotherapeutic agent targeting G2
phase would be more effective than an agent arresting cells in G1 phase.
Recent work suggests that BRCA1 regulates the activity of MRN complex.
Since FOXM1 regulates BRCA2 and a member of MRN complex, it could be possible
that FOXM1 also regulates BRCA1. BRCA1 has been implicated in the transcription
of several genes in response to DNA damage, such as p21Cip1 and GADD45
(Venkitaraman 2001). This could be another link between FOXM1 and DNA repair.
Protein assembly at sites of damage is important for the DNA damage
response cascade and DNA repair. It would be interested to investigate deeper
whether FOXM1 affects protein assembly.
Previous studies showed that reduced FOXM1 expression significantly
diminished DNA replication and mitosis in tumour cells. The current study suggests
that inhibition of FOXM levels lead to defective DNA repair. Thereby, it would
beneficial to study FOXM1 inhibitor, thiostrepton, and investigate whether it inhibits of
ATM activation, reduces NBS1 levels as well as abolishes DNA repair and induces
cell cycle arrest and cell death.
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CHAPTER 6 FINAL DISCUSSION
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Breast cancer is the most common cancer in the UK. Patients are treated
according to their receptor status. ER/PR positive receptors breast cancer patients
are treated with hormonal therapy and tamoxifen is the main agent given. However,
30% of patients that initially respond to tamoxifen become resistant. Hormone
receptor (HR) negative breast cancer patients only respond to chemotherapy. A wide
range of chemotherapeutic agents is used for solid tumours, but these agents are not
specific to types of cancer. Based on the fact that there is no biomarker for HR
negative patients, treatment administrated are not specific to these breast cancers. In
addition to the lack of specificity, patients treated with chemotherapeutic agents
become resistant. Therefore, this information raises the urgent need to identify new
targets involved in hormonal and chemotherapy drugs resistance, and to develop
targeted agents.
FOXM1 is a master transcription factor involved in the regulation of cell
proliferation, cell survival, angiogenesis and metastasis. FOXM1 is a proliferation
specific factor largely expressed in developing embryos and observed at very low
levels in adults. The upregulation of FOXM1 has been widely observed in several
types of cancer, including breast cancer. The last past years, studies showed FOXM1
as an attractive target for anti-cancer drugs due to its specific expression in actively
proliferating cells, especially cancer cells, and its role in cell cycle proteins regulation.
Recent evidences raise FOXM1 as a novel target for prevention of drug resistance.
In this thesis, I have documented FOXM1 involvement in anti-estrogen
resistance. A recent study demonstrated elevated FOXM1 mRNA and protein levels
in cisplatin resistant breast cancer compared to cisplatin sensitive. I hypothesized
that FOXM1 could have an important role conferring tamoxifen resistance through the
upregulation of cell cycle target genes. In addition, FOXM1 has been recently linked
with DNA repair mechanism through the regulation of BRCA2 and XRCC1 (Tan,
Raychaudhuri et al. 2007). Therefore, I suggested that FOXM1 could also be
involved in epirubicin resistance through the upregulation of DNA repair target genes.
If FOXM1 were found important in conferring both hormone receptor positive
and negative drug resistances, FOXM1 inhibitors could be used to circumvent any
types of breast cancer resistance. Indeed, I have detailed the mechanism of FOXM1
transcriptional regulation by ERα in tamoxifen sensitive breast cancer cells. Taken
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together with a recent study, FOXM1 is regulated in a positive feedback loop with
ERα that can be used to stop and perhaps kill breast cancer cells with the ERα
antagonist, tamoxifen. I also unravelled that FOXM1 mRNA is elevated in tamoxifen
resistant cells related to tamoxifen sensitive cells, suggesting that FOXM1 is
regulated at transcriptional level in tamoxifen resistant cells. My data indicate that
FOXM1 is the major proliferation factor in tamoxifen resistant cells and that tamoxifen
has no effect on the viability of these cells. I suggest that the lack of response to
tamoxifen is likely to be due to deregulation of the ER-FOXM1 feedback loop and
upregulation of the cyclin D1-FOXM1 loop. Furthermore, my studies showed that
targeting FOXM1 in these cells can resensitise tamoxifen resistant cells to G1 growth
arrest tamoxifen-induced. Indeed, FOXM1 regulates a large range of cell cycle
regulators but this study hints that the targeting of FOXM1 results in a significant cell
growth inhibition.
Pursuing the study, I found that FOXM1 is also upregulated in epirubicin
resistant cells compared to epirubicin sensitive breast cancer cells. I unravelled a
differential regulation of FOXM1 in sensitive and resistant cells. While FOXM1 is
transcriptionally repressed by p53 via E2F1 in epirubicin sensitive cells, p53 is lost
and FOXM1 is regulated by ATM through E2F1 in epirubicin resistant cells. A recent
study has shown that FOXM1 phosphorylation and stabilization after DNA damage
through the checkpoint kinase CHK2 promotes the transcriptional expression of DNA
repair proteins, BRCA2 and XRCC1 (Anzick, Kononen et al. 1997, Tan,
Raychaudhuri et al. 2007). This report identified NBS1 as a novel DNA repair FOXM1
target gene. In addition, this study showed that FOXM1 is a crucial component of the
DNA damage response by activating ATM indirectly. In response to DNA damage,
the MRE11/RAD50/NBS1 complex regulates the activation of ATM kinase. Here I
showed that FOXM1 is required to regulate a member of the MRE11/RAD50/NBS1
complex, which is likely to promote ATM activation. ATM is the key activator of the
double and single strand DNA repair pathways. Therefore, FOXM1 involvement in
DNA repair was investigated and showed that FOXM1 knock-down completely
abrogated DNA repair. FOXM1 is well-described to be required for cell survival but
this study showed that FOXM1 is also essential for DNA repair and that targeting
FOXM1 lead to DNA repair default and can induce cell death.
182
Drug resistance raises significant clinical challenges. The mechanisms by
which cells acquire drug resistance are multiple. In the present study we have
demonstrated for the first time that FOXM1 possesses a crucial role in tamoxifen and
epirubicin resistance in breast cancer cells through enhancing cell proliferation and
DNA-damage repair pathways. Several observations suggest that FOXM1
expression is an important determinant of tamoxifen and epirubicin sensitivity and
resistance. Following drug treatment, FOXM1 was downregulated in the sensitive
MCF-7 cells while the resistant MCF-7 cells showed an up-regulation of both FOXM1
mRNA and protein expression levels. Moreover, expression of the constitutively
active ΔN-FOXM1 was sufficient to confer resistance to the cell cycle arrest OHT-
and epirubicin-induced whereas the depletion of FOXM1 through siRNA knockdown
reversed this effect.
These observations may have implications in the development of a treatment
for tamoxifen and epirubicin resistant patients, suggesting it would be more efficient
to target a key oncogene such as FOXM1, rather than targeting a proliferative gene
or DNA repair machinery, where potential compensatory mechanisms could occur.
So far, the FOXM1 inhibitor, Thiostrepton, has only been approved by the FDA
for the treatment of topical bacterial infection in cats and dogs. However,
Thiostrepton has now been largely tested in different fibroblast, breast, colon; lung
cancer cells (Bhat, Zipfel et al. 2008, Gartel 2008, Bhat, Halasi et al. 2009). Studies
reveal that Thiostrepton inhibits FOXM1 expression, but not the expression of other
members of the Forkhead box family. In addition, Thiostrepton inhibits the growth and
induces apoptosis in human cancer cell lines of different origin (Bhat, Halasi et al.
2009). The anti-cancer properties of Thiostrepton in breast cancer were not only
investigated in vitro, but also in xenograft mouse models of breast cancer in vivo. The
encapsulation of Thiostrepton enhanced its solubility and accumulation into tumour
sites. Micelle-thiostrepton nanoparticules reduces tumour growth rate with the
suppression of FOXM1 protein and induction of cell death (Wang and Gartel 2011) .
Moreover, the inhibition of FOXM1 and co-treatment with anti-proliferative or
DNA-damaging agents may be hypothesized to enhance therapeutic response.
FOXM1 inactivation has already been tested for overcoming cisplatin resistance in
breast cancer cells. SRB proliferative assays indicated that combination of cisplatin
183
and Thiostrepton showed synergistic effect on cell death rate in cells resistant to
cisplatin, and that inhibition of FOXM1 is able to circumvent cisplatin resistance in
breast cancer cells (Kwok, Myatt et al. 2008). The combination of Thiostrepton and
Bortezomib (proteasome inhibitor) was also investigated and showed a similar
synergistic effect on the induction of apoptosis in different cancer cells (Pandit and
Gartel 2011).
The mechanisms by which FOXM1 activity and expression are upregulated in
tamoxifen and epirubicin resistant cells require further investigation. However this
study and recent publications suggest a positive feedback loop between FOXM1 with
cyclin D1 and B-myb in tamoxifen resistant cells and with ATM in epirubicin resistant
cells. These observations may also have implications in the development of new co-
treatments. Cyclin D1 overexpression has been found in many cancer and correlate
with the lack of response to tamoxifen in breast cancer. A study showed that
inhibition of cyclin D1 expression by cyclin D1 shRNAs in human oral squamous cell
carcinoma cells is associated with increased cisplatin chemosensitivity (Zhou, Zhang
et al. 2009). DNA repair pathways can enable tumour cells to survive DNA damage
that is induced by chemotherapeutic treatments; therefore, inhibitors of specific DNA
repair pathways might be efficient when used in combination with DNA-damaging
chemotherapeutic drugs. The combination of ATM inhibitor with IR and doxorubicin
has been tested and showed a significant increase in the sensitivity of lung cancer
cells to the cytotoxic effect of these treatments (Shaheen, Znojek et al. 2011).
Patients with defect in one repair pathway could potentially be treated with the
inhibitor of the other pathway and benefit from maximum treatment outcome with
minimal toxicity. This thesis and a recent study suggest that FOXM1 plays a key role
in homologous recombination (Monteiro, Khongkow et al. 2012). Therefore, targeting
a critical protein in non-homologous end joining such as DNA-PK in combination with
FOXM1 inhibitor might abrogate any possible repair and increase the sensitivity of
resistant breast cancer cells to chemotherapies.
The novel thiazole antibiotic Thiostrepton is a potential pre-clinical candidate
that should be further studied in co-treatment with key inhibitors for overcoming
breast cancer drug resistance.
184
Figure 6.1 Thiostrepton as a potential candidate to overcome endocrine and chemotherapy resistance in breast cancer. Tamoxifen treatment (OHT) inhibits FOXM1 expression via ERα and FOXM1 functions in OHT-sensitive breast cancer cells, while OHT induces a range of key proteins regulated in a feedback loop with FOXM1 allowing cell survival and drug resistance in OHT-insensitive breast cancer cells. Epirubicin treatment represses FOXM1 expression via p53 upregulation in epirubicin sensitive cells, while FOXM1 is regulated in a positive feedback loop with ATM preventing DNA damage accumulation and cell death. The use of Thiostrepton in combination with these treatments could break the positive feedback loop regulating FOXM1 and its targets, and overcome Tamoxifen and Epirubicin resistance.
185
CHAPTER 7 SUPPLEMENTAL DATA
186
Figure S.D.7.1. Schematic representation of the Apa I FOXM1 construct, showing the wild-type ERE, and three mutants ERE (mERE) sequences (mutant analysed by Demetra Constantinidou).
Figure S.D.7.2. ERα induces the transcriptional activity of the human FOXM1 gene through an ERE proximal to the transcription start site (experiment performed by Demetra Constantinidou). COS-1 cells were transfected with pGL3-F Length, pGL3-ApaI) or pGL3-ERE promoter constructs, together with increasing amounts (0, 0.1, 1, 10, and 20 ng) of ERα expression vector.
187
Figure S.D.7.3 Ectopic expression of FOXM1 reduces MCF-7 cells sensitivity to cell death (Experiment performed by Julia K. Langer). MCF-7 cells wild-type, stably transfected with pcDNA3 or with FOXM1 were treated with 1μmol/L of epirubicin for 0, 4, 8, 16 24 and 48 h. At indicated times, cells were harvested for western blot analysis to determine the protein expression of FOXM1, cleaved PARP, indicator of apoptosis, and β-tubulin.
188
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